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Porcine circovirus type 2 recombinant cap protein and subunit vaccine

A technology of porcine circovirus and protein, which is applied in the field of molecular biology, can solve the problems of no prokaryotic expression of Cap protein immunoprotection, no Cap protein immunogenicity analysis, etc., and achieves low cost, simple preparation process, and high antigen purity Effect

Active Publication Date: 2011-09-07
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Vaccine research mainly focuses on the expression of Cap protein with different expression systems, but there is no immunogenicity analysis of different fragments of Cap protein, and there is no report on the immune protection effect of prokaryotic expression of Cap protein and its use in vaccine research

Method used

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  • Porcine circovirus type 2 recombinant cap protein and subunit vaccine
  • Porcine circovirus type 2 recombinant cap protein and subunit vaccine
  • Porcine circovirus type 2 recombinant cap protein and subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 PCR amplification of Cap protein a-e gene fragment and construction of prokaryotic expression plasmid

[0039] 1.1 Primer design

[0040] Primers were designed according to the PCV2 SH strain gene sequence (AY686763), and the upstream and downstream primers were respectively introduced into BamH I and Xho I sites to amplify ORF2 in 51-150, 51-200, 51-234, 101-200 and 101-233aa respectively. Gene fragments, and named Cap-a (SEQ ID NO.8), Cap-b (SEQ ID NO.9), Cap-c (SEQ ID NO.2), Cap-d (SEQ ID NO.10), Cap-e (SEQ ID NO. 11). The primer sequences are as follows:

[0041] Cap51F AA GGATCC CGCACCATCGGTTATAC (SEQ ID NO. 3)

[0042] Cap101F CC GGATCC GTTAAGGTTGAATTCTG (SEQ ID NO. 5)

[0043] Cap150R TAT CTCGAG TATGGTATGGCGGGAGG (SEQ ID NO. 6)

[0044] Cap200R TAA CTCGAG TGCCGAGGCCTACA (SEQ ID NO. 7)

[0045] Cap233R CTT CTCGAG TCACTTAGGGTTAAGTGGG (SEQ ID NO. 4)

[0046] The above primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

...

Embodiment 2

[0051] Example 2 Construction of recombinant expression strain BL21-Cap-a / b / c / d / e

[0052] 2.1 Transformation of competent Escherichia coli BL-21

[0053] In Example 1, the correct recombinant plasmid was sequenced to transform Escherichia coli BL-21 (NEB), and the recombinant expression strains BL21-Cap-a, BL21-Cap-b, BL21-Cap-c, BL21-Cap-d, BL21-Cap-e, meanwhile, the empty plasmid pET-32a was transformed into competent Escherichia coli BL-21 in the same way.

[0054] 2.2 Optimization of recombinant protein expression conditions

[0055] Pick the recombinant expression strain BL21-Cap-a / b / c / d / e and Escherichia coli BL-21 containing the empty plasmid pET-32a and inoculate them in LB liquid medium containing ampicillin, overnight at 37°C Shake culture.

[0056] (1) Inoculate 10 μL of the overnight cultured bacterial solution into 3 ml of LB liquid medium containing ampicillin (50 μg / ml), shake and culture at 200 rpm at 37°C for about 3 hours, and make the OD 600 When it rea...

Embodiment 3

[0066] Example 3 Mass expression, purification and antigenic identification of fusion protein

[0067] 3.1 Mass expression and purification of fusion protein

[0068] According to the best induction expression condition explored in Example 2, induce the expression of 500ml of genetically engineered recombinant bacteria, collect the thalline by centrifugation, add 5ml of PBS to resuspend the thallus for every 100ml of bacterium liquid, centrifuge after ultrasonic lysis, separate the supernatant and inclusion bodies, and include The body was resuspended with an equal volume of PBS, and the inclusion body was washed and purified with the inclusion body washing solution. The steps are as follows:

[0069] (1) Centrifuge at 8000rpm for 15min, resuspend the pellet in inclusion body washing solution I (50mM Tris-HCl; 100mM NaCl; 10mM EDTA; 1% Triton X-100), 4°C for 1-2h;

[0070] (2) Centrifuge at 8000rpm for 15min, resuspend the pellet in inclusion body washing solution II (50mM Tr...

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Abstract

The invention belongs to the field of molecular biology, and discloses a porcine circovirus type 2 recombinant cap protein and a subunit vaccine. The porcine circovirus type 2 cap protein expressed by recombinant Escherichia coli is obtained by steps of cloning a porcine circovirus type 2 cap protein in a nuclear localization signal area of which the N terminal is cut and which is rich in arginine into a prokaryotic expression vector to obtain a recombinant expression vector, transfecting the recombinant expression vector into Escherichia coli BL21(DE3), and expressing by using the recombinant Escherichia coli BL21(DE3). Tests prove that the constructed recombinant strain expresses a foreign protein stably. When the subunit vaccine is prepared from the expressed recombinant protein, an antigen has high purity and safety, does not have pathogenicity on animals such as pigs and the like, and passes safety evaluation easily.

Description

technical field [0001] The invention belongs to the field of molecular biology and relates to a porcine circovirus type 2 recombinant Cap protein and a subunit vaccine. technical background [0002] The Escherichia coli expression system is currently a relatively mature expression system, which has the advantages of simple operation, safety and non-toxicity, and high expression of foreign proteins, and has been successfully used in the production of various proteins. Cap protein is about 30kD capsid protein encoded by the ORF2 gene of porcine circovirus type 2 (PCV2), which contains multiple antigenic epitopes and neutralizing antigenic epitopes, and has been recognized as the main immune protection of PCV2. antigen. Vaccine research mainly focuses on the expression of Cap protein with different expression systems, but there is no immunogenicity analysis of different fragments of Cap protein, and there is no report on the immune protection effect of prokaryotic expression o...

Claims

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Application Information

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IPC IPC(8): C07K14/01C12N15/70C12N1/21A61K39/12A61P31/20C12R1/19
Inventor 姜平李文良王先炜李玉峰
Owner NANJING AGRICULTURAL UNIVERSITY
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