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Expressed rabies virus glycoprotein optimized through gene modification and monoclonal antibody and application thereof

A rabies virus and monoclonal antibody technology, applied in the field of immunology and biology, to achieve the effect of increasing immunogenicity and facilitating screening

Inactive Publication Date: 2015-09-23
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for different genes and homologous genes from different species (differences in gene sequence and properties, etc.), how to modify the gene, determine the modification site and the number of modifications, so that it matches the partial tropism of the expressing bacteria, so as to improve protein expression. how to culture during the expression process, how to ensure the dissolved oxygen required for protein expression, how to avoid contamination, the amount and time of methanol induction, the control of protein expression time, that is, the protein harvest time, and the method of sterility test There are large differences in the determination of protein content, activity, etc., and it is necessary to form a stable and complete technical solution to fundamentally solve the problem of high-efficiency expression and cost of G protein

Method used

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  • Expressed rabies virus glycoprotein optimized through gene modification and monoclonal antibody and application thereof
  • Expressed rabies virus glycoprotein optimized through gene modification and monoclonal antibody and application thereof
  • Expressed rabies virus glycoprotein optimized through gene modification and monoclonal antibody and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 Obtaining of Modified G Protein Gene G3cDNA

[0040] 1. Use bioinformatics software to analyze the sequence of the glycoprotein extramembrane region of rabies virus HEP-Flury strain, and select the 100-307 amino acids in the dominant segment of the antigenic epitope (the antigenic epitope analysis of RV GP is shown in the attached figure 1 -A), modify the amino acid codon according to the codon preference of Escherichia coli, the sequence is shown in the attached figure 1 As shown in -B, BamHI and HindIII restriction sites were introduced at the 5' end and 3' end, respectively, with a theoretical length of 648 bp. The sequence was synthesized by Shanghai Yingjun Biotechnology Co., Ltd. and named G3.

[0041] 2, figure 2 As a result of gel electrophoresis of the G3PCR product, the amplified target gene fragment G3 double digestion purified product (swimming lane 3) is consistent with the expectation, which is 648bp (including double restriction sites and prot...

Embodiment 2

[0042] Example 2 Construction of pET32-G3 recombinant plasmid

[0043] 1. The PCR amplification product of the G3 gene was digested by BamH I and Hind III, and after recovery, it was inserted into the BamHI and Hind III double digestion window of the pET-32a(+) vector to obtain the recombinant plasmid of the G3 fusion protein gene fused with the His tag , build process like image 3 shown; image 3 The amplified product of the G3 gene was double-digested with BamH I and Hind III, and inserted into the BamH I and Hind III double-digestion window of pET-32a(+), to obtain a recombinant plasmid containing the XXX-G3 fusion protein gene.

[0044] 2. Transform Escherichia coli BL21 with the recombinant plasmid, spread the Escherichia coli BL21 on the LB plate containing ampicillin, overnight at 37°C, pick the plasmid transformed bacteria for amplification the next day, extract the plasmid DNA for enzyme digestion identification, and On 1.0% agarose gel electrophoresis, it can be s...

Embodiment 3

[0046] Example 3 Induced expression of G3 fusion protein fused with His tag

[0047] 1. Pick a single colony identified to contain the recombinant expression plasmid pET32-G3 in the Amp-containing + (100 μg / mL) in LB liquid medium, culture overnight at 37°C. Take the above-mentioned culture bacteria and inoculate them into fresh Amp-containing + (100μg / mL) 2×YT culture solution, cultivated at 37°C until the OD600 value was 0.5, then added IPTG to a final concentration of 1.0mmol / L to induce the expression of GP, and continued to cultivate at 37°C for 4h, and the induced product was 12000r After centrifuging for 1 min at 100 Å / min, collect the supernatant and cells respectively, add the same volume of 2×SDS-PAGE Loading Buffer to the supernatant, add 200 μL 1×SDS-PAGE Loading Buffer to the cells, freeze and thaw three times, boil for 10 min, Centrifuge at 12000r / min for 10min to remove large protein fragments, and take the supernatant for SDS-PAGE protein electrophoresis imme...

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Abstract

The invention discloses a modified G protein gene G3 of rabies virus. The nucleotide sequence of the G protein gene G3 is as shown by SEQ ID NO.1; the amino acid sequence of modified rabies virus G protein obtained through expression of the G protein gene G3 is as shown by SEQ ID NO.2; the rabies virus is a rabies virus HEP-Flury strain. Expression and preparation of a monoclonal antibody are performed by using the modified G protein gene G3 of the rabies virus HEP-Flury strain to construct a recombinant plasmid carrier. The monoclonal antibody can specifically recognize rabies virus glycoprotein and rabies virus, can be applied to indirect ELISA, Western-blotting tests and indirect immunofluorescence assay, can be used for detecting rabies virus and has good sensitivity and specificity.

Description

technical field [0001] The invention belongs to the field of immunology and biotechnology. More specifically, it relates to a rabies virus glycoprotein after genetic modification to optimize expression, its monoclonal antibody and application. Background technique [0002] Rabies is a severe infectious disease caused by rabies virus (rabies virus, RV), which is difficult to treat once it occurs. Accurate, timely and rapid diagnosis is the premise of clinical treatment, the main means of immune monitoring and epidemiological investigation, and the key to effective control of rabies. The glycoprotein (glycoprotein, GP) of rabies virus, that is, G protein, is one of the main structural proteins of rabies virus, which is closely related to the infection and immunity of the virus, and is the main protein that induces neutralizing antibodies. Viral vaccines are basically the same. GP consists of 524 amino acids, and the first 19 amino acids constitute a hydrophobic signal pepti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/47C07K14/145C07K16/10G01N33/577G01N33/569C12N15/70
Inventor 郭霄峰张戴婷郑佳琳阳佑天王怡飞
Owner SOUTH CHINA AGRI UNIV
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