1. The rabbit hemorrhagic disease virus VP60 recombinant antigen with the pasteurella PlpE epitope embedded at the double sites is characterized in that the recombinant antigen comprises rabbit hemorrhagic disease virus VP60 protein, and the N end and the C end of the recombinant antigen are respectively connected with a PlpE up polypeptide fragment and a PlpE down polypeptide fragment; wherein the PlpE up polypeptide fragment and the PlpE down polypeptide fragment are derived from Pasteurella PlpE protein epitope.

2. The rabbit hemorrhagic disease virus VP60 recombinant antigen with the double-site chimeric Pasteurella pasteurella PlpE epitope as claimed in claim 1, wherein the amino acid sequences of the PlpE epitope embedded at the N-terminal and C-terminal of the VP60 protein are SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

3. The recombinant antigen of rabbit hemorrhagic disease virus VP60 chimeric with Pasteurella pasteurella PlpE epitope at two sites of claim 1, wherein the nucleotide sequences of the PlpE epitope embedded at the N-terminal and the C-terminal of the VP60 protein are SEQ ID NO. 3 and SEQ ID NO. 4, respectively.

4. The rabbit hemorrhagic disease virus VP60 recombinant antigen with the Pasteurella multocida PlpE epitope embedded in the double sites of claim 1, wherein the amino acid sequence of the rabbit hemorrhagic disease virus VP60 recombinant antigen with the Pasteurella multocida PlpE epitope embedded in the double sites is SEQ ID NO: 5.

5. The rabbit hemorrhagic disease virus VP60 recombinant antigen with the double-site chimeric Pasteurella multocida PlpE epitope as claimed in claim 1, wherein the nucleotide sequence of the rabbit hemorrhagic disease virus VP60 recombinant antigen encoding the double-site chimeric Pasteurella multocida PlpE epitope is SEQ ID NO 6.

6. An expression vector for recombinant antigen, which is used for expressing rabbit hemorrhagic disease virus VP60 recombinant antigen with a double-site chimeric Pasteurella PlpE epitope and comprises the nucleotide sequence of claim 5.

7. A method for preparing the rabbit hemorrhagic disease virus VP60 recombinant antigen chimeric with Pasteurella pasteurella PlpE epitope at two sites as described in claim 1, which comprises the following steps:

(1) amplifying DNA sequences corresponding to 26 th to 50 th amino acids and 51 st to 95 th amino acids of Pasteurella PlpE by PCR;

(2) sequentially and respectively connecting the PCR products obtained in the step (1) to the 5 'end and the 3' end of the VP60 gene, and connecting the recombinant gene to a pFastBac1 cloning site to obtain a recombinant shuttle plasmid vector;

(3) transforming the recombinant shuttle vector obtained in the step (2) into DH10Bac host bacteria, and obtaining baculovirus plasmids through the transposition of the bacteria;

(4) after the recombinant baculovirus plasmid is transferred into sf9 cells, rabbit hemorrhagic disease virus VP60 recombinant protein antigen embedded with Pasteurella PlpE epitope is obtained.

8. The use of the rabbit hemorrhagic disease virus VP60 recombinant antigen with the pasteurella PlpE epitope embedded in the double sites of claim 1 in the preparation of vaccines for preventing rabbit viral hemorrhagic disease and rabbit pasteurellosis.

Background

Rabbit hemorrhagic disease, commonly known as Rabbit plague, is a highly contagious, high-morbidity and highly lethal disease caused by Rabbit Hemorrhagic Disease Virus (RHDV), and mainly occurs in adult rabbits with the age of more than 2 months. The RHDV capsid protein VP60 consists of 579 amino acids, which is the most basic unit of viral capsid composition. The capsid protein VP60 is an immunoprotective antigen of the virus, is directly related to the induction of anti-infection immunity of the organism, and independently expresses the VP60 protein to be capable of self-polymerizing into virus-like particles, so that the vaccine prepared from the virus-like particles can be used for preventing rabbit viral hemorrhagic disease.

Pasteurellosis (Pasteurellosis) in rabbits is mainly caused by pasteurella multocida ((R))Pasteurella multocida) Causing a major bacterial blight which is harmful to rabbit industry. Rabbits of various ages and varieties are susceptible to pasteurella multocida, and rabbits of 2-6 months are particularly susceptible to the disease, which is one of the main epidemic diseases causing death of rabbits of 9 weeks to 6 months. At present, protective antigens such as PlpE and the like are reported, and animal experiments show good immunoprotection efficacy.

At present, the bivalent inactivated vaccine for the rabbit viral hemorrhagic disease and the rabbit pasteurellosis is used for simultaneously preventing and controlling the rabbit viral hemorrhagic disease and the rabbit pasteurellosis. However, the vaccine has the problems of high production cost and the like, and the immune protection effect on the pasteurellosis is not ideal. The preparation process of the traditional bivalent inactivated vaccine for the rabbit hemorrhagic disease and the rabbit pasteurellosis comprises the steps of preparing the rabbit hemorrhagic disease and the rabbit pasteurellosis inactivated vaccine respectively, and then carrying out physical mixing to prepare the combined vaccine. The production process of the bivalent vaccine needs to go through the production process of two vaccines, two workers, materials, water and electricity and the like are consumed, and the production cost is increased. Although there is a trend of using genetic engineering subunit vaccines (using VP60 and PlpE as antigens) to replace traditional bacterial or viral inactivated vaccines in recent years, the current production process of genetic engineering bivalent subunit vaccines still needs to go through the process of preparing single vaccine separately and then mixing physically, and the production cost is not reduced significantly. Therefore, the scheme of forming a new antigen by chimeric connection of different antigens and epitopes thereof becomes a new combined vaccine development direction, which simplifies the production process of the combined vaccine and effectively reduces the production cost.

The invention aims to display the epitope of the pasteurella protective antigen PlpE on rabbit hemorrhagic disease virus VP60 in a chimeric way, thereby obtaining a new antigen required by the rabbit viral hemorrhagic disease and rabbit pasteurella disease bigeminal subunit vaccine.

Disclosure of Invention

The technical problem solved by the invention is as follows: the recombinant protein antigen VP60 of rabbit hemorrhagic disease virus embedded with the pasteurella protective antigen PlpE epitope is constructed to obtain the recombinant protein antigen which can produce the rabbit hemorrhagic disease virus and pasteurella with immunoprotection only by consuming one part of manual work, thereby being applied to the preparation of the bigeminal vaccine of rabbit viral hemorrhagic disease and rabbit pasteurella disease

In order to solve the technical problems, the invention provides a rabbit hemorrhagic disease virus VP60 recombinant antigen with a pasteurella PlpE epitope embedded at a double site, wherein the recombinant antigen comprises rabbit hemorrhagic disease virus VP60 protein, and the N end and the C end of the recombinant antigen are respectively connected with a PlpE up polypeptide fragment and a PlpE down polypeptide fragment; wherein the PlpE up polypeptide fragment and the PlpE down polypeptide fragment are derived from Pasteurella PlpE protein epitope.

The amino acid sequences of the PlpE epitope embedded at the N end and the C end of the VP60 protein are SEQ ID NO. 1 and SEQ ID NO. 2 respectively.

The nucleotide sequences of the PlpE epitope embedded at the N end and the C end of the VP60 protein are SEQ ID NO. 3 and SEQ ID NO. 4 respectively.

The amino acid sequence of the rabbit hemorrhagic disease virus VP60 recombinant antigen with the pasteurella PlpE epitope embedded at the double sites is SEQ ID NO. 5; the nucleotide sequence of the rabbit hemorrhagic disease virus VP60 recombinant antigen for coding the double-site chimeric Pasteurella pneumophila PlpE epitope is SEQ ID NO. 6.

In addition, an expression vector for a recombinant antigen for expressing a rabbit hemorrhagic disease virus VP60 recombinant antigen having an epitope of Pasteurella pasteurella PlpE antigen chimeric at two sites, comprising the nucleotide sequence of claim 7 is provided.

The invention relates to a preparation method of a rabbit hemorrhagic disease virus VP60 recombinant antigen with a Pasteurella pasteurella PlpE epitope embedded at double sites, which comprises the following steps:

(1) amplifying DNA sequences corresponding to 26 th to 50 th amino acids and 51 st to 95 th amino acids of Pasteurella PlpE by PCR;

(2) sequentially and respectively connecting the PCR products obtained in the step (1) to the 5 'end and the 3' end of the VP60 gene, and connecting the recombinant gene to a pFastBac1 cloning site to obtain a recombinant shuttle plasmid vector;

(3) transforming the recombinant shuttle vector obtained in the step (2) into DH10Bac host bacteria, and obtaining baculovirus plasmids through the transposition of the bacteria;

(4) after the recombinant baculovirus plasmid is transferred into sf9 cells, rabbit hemorrhagic disease virus VP60 recombinant protein antigen embedded with Pasteurella PlpE epitope is obtained.

The rabbit hemorrhagic disease virus VP60 recombinant antigen with the pasteurella PlpE epitope embedded in the double sites is applied to the preparation of vaccines for preventing rabbit viral hemorrhagic disease and rabbit pasteurellosis.

The invention has the beneficial effects that: compared with the prior art, although the rabbit viral hemorrhagic disease and the rabbit pasteurella combined inactivated vaccine or the genetic engineering subunit vaccine are adopted to simultaneously prevent and control the rabbit viral hemorrhagic disease and the rabbit pasteurella disease in the prior art, the traditional preparation process of the combined vaccine is to prepare two vaccines respectively and then physically mix the two vaccines, the production process of the combined vaccine needs to go through the production process of the two vaccines, two manual work, materials, water and electricity and the like are consumed, the production cost is increased, and the risk of mutual interference of different components is brought. Although the rabbit hemorrhagic disease virus structural protein VP60 theoretically has the capability of displaying foreign epitopes at the N-terminal and the C-terminal, great difficulty is faced in displaying foreign epitopes (particularly bacterial epitopes with relatively far relativity) as much as possible and ensuring that the self-epitopes are not influenced (the VP60 protein can only display foreign animal virus epitopes of about 40 amino acids at the longest at the N-terminal and the C-terminal under the current technical conditions). On the basis of analyzing the composition of the Pasteurella multocida protective antigen PlpE epitope, a section of region (total length of 70 amino acids) in which the PlpE epitope is intensively distributed is displayed at the N end and the C end of the rabbit hemorrhagic disease virus VP60 protein in a two-section chimeric way, and the recombinant antigen is unexpectedly found to still have hemagglutination activity (namely, virus-like particles can be formed, and the neutralizing epitope of the rabbit hemorrhagic disease virus is not influenced). The immune protective force experiment result shows that the recombinant antigen has immune protective effect on pasteurella and rabbit hemorrhagic disease virus.

In a word, the invention obtains the recombinant antigen with immunoprotection effect on rabbit hemorrhagic disease virus and pasteurellosis by constructing the rabbit hemorrhagic disease virus Vp60 recombinant protein embedded with the pasteurellosis protective antigen PlpE epitope, and provides a foundation for later-stage production of lower-cost rabbit viral hemorrhagic disease and rabbit pasteurellosis vaccines.

Drawings

FIG. 1 shows the prediction of the ability of the amino acid residues of PlpE to form B cell epitopes (using the online software Bepipred-2.0 (http:// tools. immuneepitope. org/bcell /), the prediction score is higher than 0.5 and the probability of forming B cell epitopes is higher);

FIG. 2 is a schematic diagram of the construction process of a recombinant shuttle plasmid vector of PlpE epitope double-site chimeric VP60 recombinant antigen;

FIG. 3 shows the expression of the PlpE epitope chimeric VP60 recombinant protein VP60-PlpE, wherein M represents a molecular size marker; VP60-PlpE is a VP60-PlpE recombinant protein (68 kDa) expressed by a baculovirus-insect cell expression system; VP60 represents the rabbit hemorrhagic disease virus VP60 protein (60 kDa) expressed by the baculovirus-insect cell expression system;

FIG. 4 shows the hemagglutination properties of VP60-PlpE after the PlpE epitope is embedded in VP60 recombinant protein, and the left picture shows the agglutination of VP60-PlpE to human type O red blood cells; right panel is control, i.e. agglutination of human O-type erythrocytes by wild-type baculovirus cultures;

FIG. 5 is a diagram showing the reaction of PlpE epitope chimeric VP60 recombinant protein VP60-PlpE with PlpE hyperimmune serum, wherein M represents molecular size markers; VP60-PlpE is VP60-PlpE recombinant protein expressed by baculovirus-insect cell expression system; VP60 represents rabbit hemorrhagic disease virus VP60 protein expressed by baculovirus-insect cell expression system;

FIG. 6 shows the specific antibody production (A) and the survival of the mouse after challenge with Pasteurella in the immunoprotection test (B)

FIG. 7 survival of rabbits after challenge with rabbit hemorrhagic disease virus (A) and Pasteurella virus (B) in immunoprotection experiments.

Detailed Description

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.

Example 1: preparation of PlpE epitope chimeric VP60 recombinant protein

(1) Plasmids, strains and culture vectors, namely plasmids pFastBac1 and pFastBac1-VP60 are constructed and stored in the laboratory;E．coli DH5 α was purchased from tokyo kezam biotechnology limited;E．coliDH10Bac is obtained from Shanghai Weidi Biotechnology GmbH, and Pasteurella multocida C51-17 is provided by laboratory preservation; sf9 insect cells were maintained and cultured in this laboratory.

Protein amino acid sequence analysis was performed using the sequence of the PlpE gene in the whole genome sequence of Pasteurella multocida strain C51-17 published by Genbank as a reference sequence. B cell epitope distribution of PlpE was determined using Bepipred-2.0 (http:// tools. immuneepitope. org/bcell /).

The B cell epitope prediction of PlpE is shown in FIG. 1, where the primary structure of PlpE comprises a total of 336 amino acid residues. The default 0.5 of the Bepipred-2.0 software was chosen as the threshold for the epitope composed of amino acid residues, and the prediction showed that the epitope for PlpE is mainly distributed at the N-terminus, especially the amino acid residue sequence from position 26 to 105. Therefore, the invention selects the N-end 26-95 amino acid to be connected to the N-end and the C-end of the rabbit hemorrhagic disease virus VP60 in a segmented manner to obtain the epitope chimeric VP60, namely the recombinant protein antigen prepared by the rabbit viral hemorrhagic disease and rabbit pasteurellosis vaccine has the amino acid sequence of SEQ ID NO. 5.

(3) Construction of recombinant bacmid the sequence of the PlpE gene of Pasteurella multocida strain C51-17 was used as a reference sequence, and the primer Plpeupf was designed: 5'-aggcatgcggtaccaagcttatgTGTAGCGGTGGTGGCGGT-3' and Plpeupr: 5'-gggctttgccctcATTTGATTGAACCGGTTGTGCT-3' is used to amplify a DNA fragment corresponding to amino acids 26 to 50 of PlpE, Plpedown F: 5'-ggcttttcttatgtcACTCCAATCAAACATCCTATGACTAATAG-3' and PlpE down R: 5'-atcctctagtacttctcgacctaTTTTTCTTGTTCTAGAGGGGCTTG-3' A DNA fragment corresponding to amino acids 51 to 95 of PlpE was amplified. The Pasteurella C51-17 strain genome is extracted and PCR amplification is carried out on the PlpE gene fragment of interest. The VP60 gene fragment was prepared by amplifying pFastBac1-VP60 plasmid vector using primers VP60F 5'-atcaaatGAGGGCAAAGCCCGCACA-3' and VP60R 5 'ggagtGACATAAGAAAAGCCATTGGTTGTG 3'. The pFastBacF 1 plasmid vector was PCR amplified using primers pFastBacF5 '-GTCGAGAAGTACTAGAGGATCATAATCAGCC-3' and pFastBacR5 '-AAGCTTGGTACCGCATGCC-3' to prepare a linearized pFastBac1 linearized vector. Gene fragments corresponding to the 26 th-50 th amino acid and the 51 th-95 th amino acid of PlpE are respectively connected to the 5 'end and the 3' end of the VP60 gene according to the operation in the instruction of a homologous recombinant cloning kit (Novozam, Nanjing), and then the positive recombinant plasmid is transferred into DH5 alpha host bacteria. And selecting positive clone PCR to identify correctly, sending the positive clone PCR to Nanjing subsection sequencing of Beijing Optimalaceae New industry Limited company to identify whether the cloned gene is complete and correctly connected to the carrier, and displaying the result of sequencing that the target gene fragment is correctly connected to the carrier. Extracting positive cloning plasmid according toE． coliProcedures in the DH10Bac competent cell Specification, transformation of recombinant plasmids intoE． coliAnd (3) obtaining positive clones in DH10Bac competent cells through blue-white screening and PCR identification, and extracting recombinant Bacmid-VP 60-PlpE. The recombinant bacmid constructed is shown in FIG. 2.

Transfection, expression and identification Bacmid-VP60-PlpE was passed through Liposomal Lipofectamin^TM3000 Sf9 insect cells transfected to logarithmic growth phase. After transfection, the cells were observed 1 time every 12 h, and when cytopathic effect was evident, the cells and supernatant were collected as a stock solution of recombinant baculovirus and stored at 4 ℃. And (3) repeatedly freezing and thawing the 1 st generation virus solution for 3 times, then inoculating Sf9 cells with the volume ratio of 1%, and carrying out passage to obtain the 2 nd generation recombinant virus. After 3 generations of virus obtained by repeating the above procedure, the recombinant baculovirus was inoculated into the suspension sf9 cells in logarithmic phase for spinner culture, and the culture was collected after about 100 hours of culture. The recombinant VP60-PlpE protein was obtained after 3 repeated freeze-thawing of the culture, and observed after SDS-PAGE and Coomassie blue staining, a band of about 68kDa was present in the sample (FIG. 3), corresponding to the theoretical predicted size of VP 60-PlpE.

Example 2 hemagglutination assay of two-site chimeric VP60-PlpE recombinant proteins

VP60-PlpE and wild type baculovirus culture were added to 50. mu.L of each well in two wells of a 50-well U-plate. Then 50. mu.L of 1% human O-type erythrocyte suspension is added into each hole, and after 30 min of action at 4 ℃, the hemagglutination condition is observed. The result shows that the VP60-PlpE recombinant protein has obvious hemagglutination (figure 4), namely, the VP60-PlpE recombinant protein is considered to still form rabbit hemorrhagic disease virus-like particles, and effectively displays the rabbit hemorrhagic disease virus neutralizing epitope.

EXAMPLE 3 reaction of the two-site chimeric VP60-PlpE recombinant protein with PlpE hyperimmune serum

The VP60-PlpE recombinant protein and VP60 recombinant protein (control) were subjected to SDS-PAGE gel electrophoresis, and then the proteins were transferred onto NC membranes. After being sealed by 5 percent skim milk, 1/500 diluted mouse anti-PlpE serum preserved in the laboratory is added, and the mixture is incubated for 1 h at 37 ℃; PBST was rinsed 5 times, 1/10000 diluted HRP-conjugated goat anti-mouse IgG was added, and incubation was performed at 37 ℃ for 1 h; PBST was rinsed 5 times and ECL color development was performed. Western blot results showed that there was a band of about 68kDa in lane VP60-PlpE (FIG. 5), but there was no band of expected size in lane VP60 as a control, indicating that the recombinant protein VP60-PlpE was able to specifically react with mouse anti-PlpE serum, i.e., the PlpE partial immunogenic fragment was successfully integrated into VP 60.

Example 4 immunoprotection assay

(1) Mouse immunoprotection experiments 20 4-6 week old female ICRs were randomly divided into 2 groups, 1 control, and another 10 mice. Experimental groups immunization of VP60-PlpE recombinant protein: after 20% volume of alumina gel was added to 200. mu.g/. mu.l of recombinant protein, the immunized mice were injected intraperitoneally at 100. mu.l each. The booster immunization after 21 days, the dose, the mode and the like are the same as those of the primary immunization. Negative control group: PBS was used instead of recombinant protein, and the adjuvant used was the same as that of the experimental group. After blood was collected from tail veins of all mice 10 days after the booster immunization, it was confirmed that high levels of specific antibodies had been produced (FIG. 6A), and then the mice were challenged subcutaneously with Pasteurella C51-17 strain at about 50CFU each (about 5 × LD 50). The survival of the mice after challenge is shown in fig. 6B, only 2 mice survived in the control group, and 8 mice survived in the experimental group. This indicates that VP60-PlpE confers immunoprotection against challenge with Pasteurella.

(2) In the rabbit immunoprotection experiment, 60 healthy and susceptible rabbits with the age of 2-3 months are selected and randomly divided into 6 groups, 2 groups of controls and 4 groups of experiments, wherein each group comprises 10 rabbits. Two experimental groups immunized against the VP60-PlpE recombinant protein group: adding 20% volume of alumina gel adjuvant into 200mg/ml recombinant protein, and injecting immunized rabbit subcutaneously, each 1 ml; two further experimental groups immunized rabbits with a physical Mixture of VP60 and PlpE recombinant protein (mix) comprising 100mg/ml each of VP60 recombinant protein and PlpE recombinant protein, after addition of 20% by volume of an alumina gel adjuvant. The remaining two groups served as negative controls: the recombinant protein was replaced with the immunophosphate using the same adjuvant as that of the experimental group. After 3 weeks of immunization, the rabbit hemorrhagic disease virus Wanfan strain was used to challenge the VP60-PlpE chimeric antigen immunization group, the physical Mixture group and a group of control groups, and the survival condition of the rabbits was observed at 100 × LD 50/rabbit for 7 days continuously. After 3 weeks of immunization, the remaining VP60-PlpE chimeric antigen immunization group, the physically mixed texture group and the other group were challenged with 100CFU (10 × MLD)/rabbit survival was observed for 7 consecutive days using Pasteurella C51-17 strain. After the rabbit hemorrhagic disease virus challenge, the survival rate of the experimental group (the immunity VP60-PlpE chimeric antigen recombinant protein group and the immunity two-protein physical mixture group) rabbits is 100%, and the control group is totally dead (the survival rate is 0/10, and the survival rate is shown in figure 7A). The survival rate of rabbits in the experimental group after pasteurella challenge was 100%, while the survival rate of the control group was only 20% (fig. 7B). The results of these experiments show that the immunization of VP60-PlpE recombinant protein can ensure that the rabbit has immunoprotection against rabbit hemorrhagic disease virus and pasteurella simultaneously, and the VP60-PlpE recombinant protein has the same protective power as the vaccine of the physical mixture of the two proteins.

Example 5 Process and time consuming comparison

Although the chimeric antigen and the two protein mixture vaccine provided by the patent have similar immune protection effects, the preparation process has obvious difference. As shown in Table 1, the chimeric antigen preparation process does not need to undergo steps of antigen purification, endotoxin removal, physical mixing and the like, the preparation time is shortened by 50%, material and water and electricity consumption of corresponding steps does not exist, the production cost is obviously reduced, and the application prospect is good.

Table 1 comparison of preparation processes

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

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Ala Thr Cys Ala Cys Cys Ala Thr Gly Cys Cys Ala Gly Ala Cys Thr

580 585 590

Thr Gly Cys Gly Thr Cys Cys Cys Ala Ala Cys Ala Thr Gly Thr Ala

595 600 605

Cys Cys Ala Thr Cys Cys Ala Ala Cys Thr Gly Gly Thr Gly Ala Cys

610 615 620

Cys Cys Thr Gly Gly Cys Cys Thr Thr Gly Thr Cys Cys Cys Cys Ala

625 630 635 640

Cys Ala Cys Thr Ala Gly Thr Cys Cys Thr Thr Ala Gly Thr Gly Thr

645 650 655

Thr Thr Ala Cys Ala Ala Cys Ala Ala Cys Cys Thr Cys Ala Thr Cys

660 665 670

Ala Ala Cys Cys Cys Gly Thr Thr Thr Gly Gly Thr Gly Gly Ala Thr

675 680 685

Cys Cys Ala Cys Cys Ala Ala Cys Gly Cys Ala Ala Thr Cys Cys Ala

690 695 700

Gly Gly Thr Gly Ala Cys Ala Gly Thr Gly Gly Ala Ala Ala Cys Gly

705 710 715 720

Ala Gly Gly Cys Cys Gly Ala Gly Thr Gly Ala Thr Gly Ala Cys Thr

725 730 735

Thr Thr Gly Ala Gly Thr Thr Cys Gly Thr Gly Ala Thr Gly Ala Thr

740 745 750

Thr Ala Gly Ala Gly Cys Cys Cys Cys Cys Thr Cys Cys Ala Gly Cys

755 760 765

Ala Ala Ala Ala Cys Thr Gly Thr Thr Gly Ala Cys Thr Cys Ala Ala

770 775 780

Thr Cys Thr Cys Ala Cys Cys Cys Gly Cys Ala Gly Gly Cys Cys Thr

785 790 795 800

Thr Cys Thr Cys Ala Cys Gly Ala Cys Cys Cys Cys Ala Gly Thr Cys

805 810 815

Cys Thr Cys Ala Cys Thr Gly Gly Thr Gly Thr Thr Gly Gly Cys Ala

820 825 830

Ala Thr Gly Ala Cys Ala Ala Cys Ala Gly Gly Thr Gly Gly Ala Ala

835 840 845

Cys Gly Gly Cys Cys Ala Ala Ala Thr Ala Gly Thr Gly Gly Gly Ala

850 855 860

Cys Thr Gly Cys Ala Ala Cys Cys Ala Gly Thr Ala Cys Cys Thr Gly

865 870 875 880

Gly Gly Gly Gly Gly Thr Thr Thr Thr Cys Cys Ala Cys Gly Thr Gly

885 890 895

Cys Ala Ala Cys Ala Gly Gly Cys Ala Cys Thr Gly Gly Ala Ala Cys

900 905 910

Cys Thr Gly Ala Ala Cys Gly Gly Cys Ala Gly Cys Ala Cys Ala Thr

915 920 925

Ala Thr Gly Gly Cys Thr Gly Gly Thr Cys Ala Ala Gly Cys Cys Cys

930 935 940

Thr Cys Gly Gly Thr Thr Thr Gly Cys Cys Gly Ala Cys Ala Thr Thr

945 950 955 960

Gly Ala Cys Cys Ala Thr Cys Gly Ala Ala Gly Ala Gly Gly Cys Ala

965 970 975

Gly Thr Gly Cys Ala Ala Gly Thr Thr Ala Thr Thr Cys Thr Gly Gly

980 985 990

Gly Ala Ala Cys Ala Ala Cys Thr Cys Cys Ala Cys Cys Ala Ala Cys

995 1000 1005

Gly Thr Gly Cys Thr Thr Cys Ala Gly Thr Thr Thr Thr Gly Gly Thr

1010 1015 1020

Ala Cys Gly Cys Thr Ala Ala Thr Gly Cys Thr Gly Gly Gly Thr Cys

1025 1030 1035 1040

Thr Gly Cys Gly Ala Thr Thr Gly Ala Cys Ala Ala Cys Cys Cys Thr

1045 1050 1055

Ala Thr Cys Thr Cys Cys Cys Ala Gly Gly Thr Thr Gly Cys Ala Cys

1060 1065 1070

Cys Ala Gly Ala Cys Gly Gly Cys Thr Thr Cys Cys Cys Thr Gly Ala

1075 1080 1085

Cys Ala Thr Gly Thr Cys Ala Thr Thr Cys Gly Thr Gly Cys Cys Cys

1090 1095 1100

Thr Thr Thr Ala Ala Cys Ala Gly Cys Cys Cys Cys Ala Ala Cys Ala

1105 1110 1115 1120

Thr Thr Cys Cys Gly Ala Cys Cys Gly Cys Gly Gly Gly Gly Thr Gly

1125 1130 1135

Gly Gly Thr Cys Gly Gly Gly Thr Thr Thr Gly Gly Thr Gly Gly Thr

1140 1145 1150

Ala Thr Thr Thr Gly Gly Ala Ala Cys Ala Gly Thr Ala Ala Cys Ala

1155 1160 1165

Ala Cys Gly Gly Thr Gly Cys Cys Cys Cys Cys Gly Cys Thr Gly Cys

1170 1175 1180

Thr Ala Cys Ala Ala Cys Thr Gly Thr Gly Cys Ala Gly Gly Cys Cys

1185 1190 1195 1200

Thr Ala Thr Gly Ala Gly Thr Thr Ala Gly Gly Thr Thr Thr Thr Gly

1205 1210 1215

Cys Cys Ala Cys Thr Gly Gly Gly Gly Cys Ala Cys Cys Ala Ala Ala

1220 1225 1230

Cys Ala Gly Cys Cys Thr Cys Cys Ala Gly Cys Cys Cys Ala Cys Cys

1235 1240 1245

Ala Cys Cys Ala Ala Cys Ala Cys Thr Thr Cys Ala Gly Gly Thr Gly

1250 1255 1260

Cys Ala Cys Ala Gly Ala Cys Thr Gly Thr Cys Gly Cys Thr Ala Ala

1265 1270 1275 1280

Gly Thr Cys Cys Ala Thr Thr Thr Ala Thr Gly Cys Cys Gly Thr Gly

1285 1290 1295

Gly Thr Ala Ala Cys Cys Gly Gly Cys Ala Cys Ala Ala Ala Cys Cys

1300 1305 1310

Ala Ala Ala Ala Thr Cys Cys Ala Ala Cys Cys Gly Gly Ala Cys Thr

1315 1320 1325

Gly Thr Thr Thr Gly Thr Gly Ala Thr Gly Gly Cys Cys Thr Cys Gly

1330 1335 1340

Gly Gly Thr Gly Thr Thr Ala Thr Cys Thr Cys Cys Ala Cys Gly Cys

1345 1350 1355 1360

Cys Ala Ala Ala Cys Gly Cys Cys Ala Gly Cys Gly Cys Cys Gly Thr

1365 1370 1375

Cys Ala Cys Ala Thr Ala Cys Ala Cys Gly Cys Cys Cys Cys Ala Ala

1380 1385 1390

Cys Cys Ala Gly Ala Cys Ala Gly Ala Ala Thr Thr Gly Thr Gly Ala

1395 1400 1405

Cys Thr Ala Cys Ala Cys Cys Cys Gly Gly Cys Ala Cys Thr Cys Cys

1410 1415 1420

Thr Gly Cys Cys Gly Cys Thr Gly Cys Ala Cys Cys Thr Gly Thr Ala

1425 1430 1435 1440

Gly Gly Thr Ala Ala Gly Ala Ala Cys Ala Cys Ala Cys Cys Cys Ala

1445 1450 1455

Thr Cys Ala Thr Gly Thr Thr Cys Gly Cys Gly Thr Cys Thr Gly Thr

1460 1465 1470

Thr Gly Thr Cys Ala Gly Gly Cys Gly Cys Ala Cys Cys Gly Gly Thr

1475 1480 1485

Gly Ala Cys Gly Thr Cys Ala Ala Cys Gly Cys Cys Gly Cys Ala Gly

1490 1495 1500

Cys Cys Gly Gly Gly Thr Cys Ala Ala Cys Cys Ala Ala Cys Gly Gly

1505 1510 1515 1520

Gly Ala Cys Cys Cys Ala Gly Thr Ala Thr Gly Gly Cys Ala Cys Gly

1525 1530 1535

Gly Gly Cys Thr Cys Cys Cys Ala Ala Cys Cys Ala Cys Thr Gly Cys

1540 1545 1550

Cys Ala Gly Thr Gly Ala Cys Ala Ala Thr Thr Gly Gly Ala Cys Thr

1555 1560 1565

Thr Thr Cys Gly Cys Thr Cys Ala Ala Cys Ala Ala Cys Thr Ala Cys

1570 1575 1580

Thr Cys Gly Thr Cys Ala Gly Cys Ala Cys Thr Cys Gly Thr Gly Cys

1585 1590 1595 1600

Cys Thr Gly Gly Gly Cys Ala Gly Thr Thr Cys Thr Thr Cys Gly Thr

1605 1610 1615

Thr Thr Gly Gly Cys Ala Gly Thr Thr Ala Ala Cys Cys Thr Thr Thr

1620 1625 1630

Gly Cys Ala Thr Cys Thr Gly Gly Thr Thr Thr Cys Ala Thr Gly Gly

1635 1640 1645

Ala Gly Ala Thr Cys Gly Gly Cys Cys Thr Ala Ala Gly Thr Gly Thr

1650 1655 1660

Gly Gly Ala Cys Gly Gly Gly Thr Ala Cys Thr Thr Thr Thr Ala Thr

1665 1670 1675 1680

Gly Cys Ala Gly Gly Ala Ala Cys Ala Gly Gly Ala Gly Cys Cys Thr

1685 1690 1695

Cys Ala Ala Cys Cys Ala Cys Gly Cys Thr Cys Ala Thr Thr Gly Ala

1700 1705 1710

Cys Thr Thr Gly Ala Cys Thr Gly Ala Ala Cys Thr Cys Ala Thr Thr

1715 1720 1725

Gly Ala Cys Gly Thr Ala Cys Gly Cys Cys Cys Cys Gly Thr Gly Gly

1730 1735 1740

Gly Ala Cys Cys Cys Ala Gly Gly Cys Cys Gly Thr Cys Cys Ala Ala

1745 1750 1755 1760

Ala Ala Gly Cys Ala Cys Ala Cys Thr Cys Gly Thr Gly Thr Thr Cys

1765 1770 1775

Ala Ala Cys Cys Thr Gly Gly Gly Gly Gly Gly Cys Ala Cys Ala Ala

1780 1785 1790

Cys Cys Ala Ala Thr Gly Gly Cys Thr Thr Thr Thr Cys Thr Thr Ala

1795 1800 1805

Thr Gly Thr Cys Thr Cys Ala Cys Thr Ala Gly Cys Ala Ala Ala Thr

1810 1815 1820

Ala Ala Ala Cys Ala Ala Cys Ala Ala Ala Thr Thr Cys Ala Ala Ala

1825 1830 1835 1840

Thr Ala Cys Cys Thr Ala Cys Ala Ala Cys Thr Cys Ala Ala Ala Ala

1845 1850 1855

Thr Thr Cys Thr Thr Thr Thr Ala Ala Ala Cys Ala Thr Ala Thr Cys

1860 1865 1870

Gly Ala Ala Gly Ala Ala Gly Ala Gly Ala Ala Ala Ala Thr Gly Ala

1875 1880 1885

Thr Ala Ala Ala Gly Gly Ala Thr Ala Cys Cys Ala Cys Ala Ala Ala

1890 1895 1900

Cys Gly Ala Thr Thr Gly Gly Thr Thr Thr Ala Ala Ala Ala Gly Thr

1905 1910 1915 1920

Thr Gly Cys Gly Thr Thr Thr Cys Cys Thr Cys Thr Thr Ala Thr Cys

1925 1930 1935

Ala Gly Ala Ala Thr Ala Cys Thr Thr Gly Thr Thr Gly Ala

1940 1945 1950