Recombinant expression and application of optimized porcine rotavirus outer capsid protein VP4

A porcine rotavirus and shell protein technology, applied in the field of genetic engineering, can solve the problems of large amount of antibiotics, few research reports and low protein expression, and achieve the effect of improving expression efficiency and strong biological activity.

Pending Publication Date: 2022-08-09
江苏三仪生物工程有限公司 +1
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, in the preparation method of porcine rotavirus outer capsid protein VP4, there are mainly the following problems: one, there are few research reports about porcine rotavirus outer capsid protein VP4 and the protein expression level is not high; two, when used for recombinant expression The amount of antibiotics used is often large, and there are hidden dangers of the spread of drug resistance genes, etc.

Method used

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  • Recombinant expression and application of optimized porcine rotavirus outer capsid protein VP4
  • Recombinant expression and application of optimized porcine rotavirus outer capsid protein VP4
  • Recombinant expression and application of optimized porcine rotavirus outer capsid protein VP4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Determination of VP4 gene sequence of porcine rotavirus outer capsid protein and its optimization method

[0039] 1. The nucleotide sequence and the amino acid sequence of the encoded protein were found in NCBI, and the porcine rotavirus outer capsid protein VP4 nucleotide sequence (SEQ ID NO.1) for synthesis and construction was obtained by screening. The literature determines the full-length sequence, open reading frame and antigenic structural functional region of porcine rotavirus outer capsid protein VP4 gene;

[0040] 2. Adjust the codon usage preference to match the highest expression profile of the target host, and increase the codon adaptation index (CAI) from 0.4 to 0.98 (CAI value 0.8-1.0 is considered good high expression);

[0041] 3. Adjust the average GC content from 35.4% to 54.7% to remove bad peaks;

[0042] 4. Remove repetitive regions in the original sequence and avoid stem-loop structures in mRNA;

[0043] 5. Modify restriction enzyme s...

Embodiment 2

[0045] Example 2: Construction of recombinant vector and expression of target protein

[0046] 1. The optimized VP4 gene sequence (SEQ ID NO.2) and pET32a plasmid (sequence such as SEQ ID NO.3) of artificial synthesis were double digested with BamH I and Xho I, and the digested product was separated by agarose gel electrophoresis. , the target band and the vector band were recovered by cutting the gel, ligated with T4 ligase, transformed into BL21(DE3) competent cells, and screened with 0.5ug / ml ampicillin to obtain a new plasmid pET32a-VP4;

[0047] 2. Pick the single colony growing on the plate, extract the plasmid after pure culture, carry out double digestion and identification of the plasmid, and send the correctly identified plasmid to Shanghai Sangong for sequencing and identification to obtain recombinant bacteria;

[0048] 3. The recombinant bacteria were shaken and cultured overnight at 37°C in LB liquid medium containing 0.5ug / ml ampicillin;

[0049] 4. Inoculate t...

Embodiment 3

[0051] Example 3: Biological Activity Verification of Recombinant Proteins

[0052] The pig farm found that 60 piglets had symptoms such as lethargy, anorexia, vomiting, and diarrhea, which were determined to be caused by rotavirus infection through pathological examination and laboratory testing. The recombinant bacteria ( Experimental group) and recombinant bacteria expressing recombinant original VP4 protein (control group) were prepared into bacteria powder for feeding experiments. Due to the adjuvant effect, adhesion properties and non-pathogenicity of the recombinant bacteria while retaining antigen specificity, it can be used as a carrier for presenting VP4 antigen protein, so it was prepared into bacterial powder for animal experiments.

[0053] The above 60 piglets were randomly divided into three groups: the experimental group, the control group and the daily feed group (without VP4 bacteria powder). The feed group was only fed with daily feed, drinking water and ot...

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Abstract

The invention belongs to the technical field of gene engineering and bioengineering, and particularly provides a preparation method of recombinant porcine rotavirus coat protein VP4 by determining gene sequence information of a main structural functional region of the porcine rotavirus coat protein VP4 by using a bioinformatics means and optimizing the sequence. According to the preparation method, optimized recombinant plasmids are expressed in an escherichia coli expression system, the method is simple, convenient and easy to operate, the expression efficiency and the biological activity are remarkably improved, the antibiotic resistance level is low, and an important basis is provided for development of porcine rotavirus oral vaccines.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular, the present invention relates to an optimized porcine rotavirus outer capsid protein VP4, its recombinant expression and its application. Background technique [0002] Porcine rotavirus (PoRV) belongs to Reoviridae, genus Rotavirus, double-stranded RNA virus, and is one of the main pathogens that cause dehydration gastroenteritis in young children and other animals. , easy to cause large-scale infection of pig farms and regardless of age. This virus can cause severe gastroenteritis in piglets, and also cause dehydration diarrhea in piglets, destroying the acid-base balance of the body. The incidence rate of piglets within 4 weeks of age can reach 80%, and the mortality rate can reach 20%. The healthy development of China's pig industry has brought huge economic losses to my country's pig industry. [0003] The known porcine rotavirus hemagglutinin antigen is determined b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/46C12N15/70C12N15/66C12P21/02C07K14/14A23K20/147A23K50/60A23K50/30
CPCC07K14/005C12N15/70A23K20/147A23K50/60A23K50/30C12N2720/12322C12N2720/12351C12N2800/22Y02P60/87
Inventor 江国托单春乔于洪敏刘艳李娟王岩刘秋晨翟宏旭
Owner 江苏三仪生物工程有限公司
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