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Antibody for inhibiting replication of PRRS virus, expression vector and therapeutic agent

A virus replication and expression vector technology, applied in the field of genetic engineering, can solve problems such as limited protective effect

Inactive Publication Date: 2019-05-03
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] To sum up, the problem with existing technologies is that although the attention of prevention and control of PRRS virus infection is focused on the development of vaccines, due to the continuous mutation of PRRS virus genes, the protective effect of existing vaccines on the disease is very limited

Method used

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  • Antibody for inhibiting replication of PRRS virus, expression vector and therapeutic agent
  • Antibody for inhibiting replication of PRRS virus, expression vector and therapeutic agent
  • Antibody for inhibiting replication of PRRS virus, expression vector and therapeutic agent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Prokaryotic expression of antibody therapeutic agent:

[0036] Through two rounds of PCR (primers and templates are as follows), the gene encoding the antibody therapeutic agent is amplified. NdeI and XhoI (purchased from NEB) were used to digest 20 μg of the pET21b prokaryotic expression vector and the product recovered by the second round of PCR, and then the two fragments were ligated at 16° C. overnight with T4 DNA ligase. Take 10 μl of the ligation product and transform it into 50 μl of expression competent cells BL21(DE3), and spread it on LB after activation AMP+ Agar plate, cultivate overnight at 37°C, pick a single colony in LB AMP+ Cultivate in liquid medium at 37°C and 220rpm for 12h, and perform sequencing analysis on the bacterial liquid identified as positive by bacterial liquid PCR. Bacteria with positive sequencing results were inoculated in LB AMP+ Cultivate in culture medium. After the bacteria grow to the logarithmic phase, use 1mM IPTG t...

Embodiment 2

[0048] Example 2 Enzyme-linked immunoassay detection of interaction between antibody therapeutic agent and Nsp9

[0049] First, Nsp9 was coated with PBS (0.01M, pH7.2) on the ELISA plate, 400ng per well, overnight at 4°C. After the ELISA plate was washed 3 times with PBS-T (PBS containing 0.5% Tween-20 by volume), it was blocked for 1 hour with a blocking solution (2.5% skimmed milk powder was dissolved in PBS-T, 200 μl / well); washed 3 times, Add antibody therapeutics with different dilution ratios to each well for reaction for 1 hour; wash 3 times, add mouse anti-His monoclonal antibody to each well and react for 1 hour; wash 3 times, add horseradish peroxidase (HRP)-labeled antibody to each well Goat anti-mouse IgG was reacted for 1 hour, washed 3 times; 100 μl of HRP substrate (TMB) was added to each well for 15 minutes, and 50 μl of 1M sulfuric acid was added to each well to stop the reaction, and the OD value of each well was read at 450 nm with a microplate reader. See ...

Embodiment 3

[0050] Example 3 Antibody Therapeutics Enter Cells

[0051] After recovery of PAMs cells, 1×10 per well 6 The density was laid in a 24-well cell culture plate, cultured for 4 hours, the old medium was discarded, and new serum-free RPMI 1640 medium was added, and the antibody therapeutic agent was added to the cell culture medium of each well at a final concentration of 5 μM, and after 5 hours of culture, Intracellular antibody therapeutics were detected using indirect immunofluorescence assays. The specific steps are as follows: discard the supernatant, wash the cells three times with PBS, add 4% paraformaldehyde, fix the cells at room temperature for 15 minutes, and permeate the membrane with 0.25% Triton X-100 at room temperature for 10 minutes. Wash 3 times with PBS, add mouse His monoclonal antibody after blocking with 1% BSA, and incubate at room temperature for 1 h. Wash 3 times with PBS, add Alexa 488-labeled goat anti-mouse fluorescent secondary antibody, placed at...

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Abstract

The present invention belongs to the technical field of genetic engineering, and discloses an antibody for inhibiting replication of a PRRS virus, an expression vector and a therapeutic agent. A nucleotide sequence of the antibody for inhibiting replication of the PRRS virus is SEQ ID NO:1. The expression vector of the present invention is capable of expressing an antibody therapeutic agent capable of entering cells, specifically binding to a PRRS virus non-structural protein Nsp9, and inhibiting proliferation of the PRRS virus in PAMs cells. The antibody therapeutic agent can be used to develop a drug for treating PRRS. The interaction of the antibody therapeutic agent with the PRRS virus Nsp9 is demonstrated by enzyme-linked immunoassay. The cells are incubated with the antibody therapeutic agent, and indirect immunofluorescence experiments are performed to demonstrate that the antibody therapeutic agent is able to enter the cells. At the same time, the antibody therapeutic agent isused for virus inhibition test to prove that the antibody therapeutic agent can inhibit the replication of the PRRS virus.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an antibody, an expression vector and a therapeutic agent for inhibiting PRRS virus replication. Background technique [0002] At present, the commonly used prior art in the industry is as follows: porcine reproductive and respiratory syndrome (Porcinereproductive and respiratory syndrome, PRRS) has another name called blue ear disease, is caused by the acute infectious disease of a kind of pig that PRRS virus (PRRSV) infects. Due to the characteristics of PRRS virus antigen variability, macrophage, antibody-dependent enhancement and persistent infection, the protective effect of existing vaccines on the disease is very limited, and there is currently no specific drug against PRRS virus. Therefore, the development of new anti-PRRS virus drugs is of great significance. [0003] PRRS virus is a single-stranded positive-strand RNA virus. The replication and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10C12N15/70A61K39/395A61P31/14
Inventor 周恩民王立珍张璐
Owner NORTHWEST A & F UNIV
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