PEDV S gene major antigen epitope serial connection recombination gene, and preparation method and application thereof

A main antigen epitope and recombinant gene technology, applied in biochemical equipment and methods, recombinant DNA technology, chemical instruments and methods, etc., can solve the problems of unstable expression, many variations in the S1 region of PEDVS protein, and poor monitoring by ELISA and assess the level of neutralizing antibodies in swine herds to achieve high-efficiency, stable expression and strong immunogenicity

Active Publication Date: 2016-09-07
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to overcome the shortcomings and deficiencies in the S1 region of PEDV S protein of most neutralizing epitopes at this stage, which have many variations, unstable expression, and the expression of other PEDV proteins, the established ELISA cannot monitor and evaluate the level of neutralizing antibodies in pigs. , the present invention provides a recombinant gene in which the main antigenic epitope of PEDV S gene is tandem

Method used

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  • PEDV S gene major antigen epitope serial connection recombination gene, and preparation method and application thereof
  • PEDV S gene major antigen epitope serial connection recombination gene, and preparation method and application thereof
  • PEDV S gene major antigen epitope serial connection recombination gene, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Synthesis of target fragments

[0040] Using the S gene of the cell-adapted mutant GDGZ12 strain isolated in our laboratory as a template, three antigenic epitopes S1 (1368-1374aa), S2 (499-638aa) and S3 (697-771aa) were selected. Linked with flexible amino acid (GGTGTGGTGGTAGC) base sequence, with XhoI and BamHI at both ends of the target fragment, this part was synthesized by Jinweizhi Biotechnology Co., Ltd., and the concatenated S gene was named S123, with a total size of 708bp. The results of PCR identification of recombinant cloned plasmids are as follows: figure 1 shown.

Embodiment 2

[0041] Example 2 Construction and identification of expression vector pET-28a-S123

[0042] 1. The vector pet-28a is connected to the target fragment S123

[0043] The prokaryotic expression vector pET-28a was double digested, and the digest reaction was carried out in a water bath at 37°C, and the digest time was 4h to 5h or overnight. After double digestion, an agarose gel was performed to recover the digestion product. After recovery, the purified and recovered target gene S123 was connected to the expression vector pET-28a, and the connection system was shown in Table 1:

[0044] Table 1 Expression vector connection system

[0045]

[0046] 2. Identification of pET-28a-S123 double enzyme digestion

[0047] Recombinant plasmid Pet-28a-S123 adopts BamHI and XhoI double-enzyme digestion identification system respectively as shown in Table 2. The digestion reaction is carried out in a 37 ℃ water bath, and the digestion time is 4h ~ 5h or overnight digestion. After double...

Embodiment 3

[0050] Example 3 Expression of target gene in Escherichia coli

[0051] Pick a single colony on the kan plate that has been identified as positive bacteria BL21 (DE3), inoculate it in 3 ml of LB medium containing kan, and set up an empty vector control group at the same time. ). The next morning, the overnight cultured bacterial solution was inoculated into 3 mL of LB medium containing kan at a ratio of 1:100, and placed in a shaker at 37 °C for 150-200 r / min and cultured until the OD600 was about 0.6-0.8. IPTG was added to The final concentration was 1.0 mmol / mL, and expression was induced for 4-6 hours. Identification by SDS-PAGE electrophoresis. At the same time, an empty vector control and a zero induction control were established. The results of recombinant plasmid expression in BL21(DE3) are shown in the figure image 3 shown.

[0052] 1 Preparation of samples for expression products

[0053] Take the bacterial samples before and after induction and set up an empty...

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Abstract

The invention discloses a PEDV S gene major antigen epitope serial connection recombination gene, and a preparation method and an application thereof, and belongs to the field of a PEDV S protein major neutralizing epitope region efficient expression method. The PEDV S gene major antigen epitope serial connection recombination gene is obtained through connecting three major antigen epitopes of a PEDV S gene in series; every two major antigen epitopes are connected through a flexible amino acid base sequence; the name is S123; and the sequence of the base sequence is shown as SEQ ID NO:1. The recombination gene contains most PEDV antigen epitopes; the selected fragments avoid a high-variation region; the expression quantity is efficient and stable; the immunogenicity is high; when the application of PEDV S gene major antigen epitope serial connection recombination protein to a PEDV indirect ELISA detection kit is used, the PEDV can be fast diagnosed; and the swinery neutralization protection can be well monitored and evaluated.

Description

technical field [0001] The invention belongs to the field of high-efficiency expression methods for the main neutralizing epitope region of PEDV S protein, and particularly relates to a recombinant gene in which the main antigenic epitopes of PEDV S gene are connected in series, and a preparation method and application thereof. Background technique [0002] Porcine epidemic diarrhea is a highly contagious enteric infectious disease caused by Porcine Epidemic Diarrhea virus and is clinically characterized by diarrhea, dehydration, vomiting and high mortality in suckling piglets. Since December 2010, Porcine Epidemic Diarrhea Virus (PEDV) has been breaking out on a large scale in my country. In 2013, it continued to sweep the American breeding industry, causing serious economic losses to the breeding industry in my country and the world. Early detection and diagnosis of the virus is extremely important, and it is extremely important to evaluate the neutralizing antibody level ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/70G01N33/68G01N33/569
CPCC07K14/005C12N15/62C12N15/70C12N2770/20022G01N33/56983G01N33/68
Inventor 贺东生王飞倪挺陈小芬焦臣鹏苏丹萍
Owner SOUTH CHINA AGRI UNIV
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