Preparation method of H7N9 pseudoviruses and applications of H7N9 pseudoviruses

A pseudovirus, avian influenza virus technology, applied in the field of biomedicine, can solve problems such as high mortality and restricting the research and development of H7N9 avian influenza

Active Publication Date: 2015-12-23
杭州庆正鸿科技有限公司
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although H7N9 avian influenza is a low-pathogenic avian influenza virus, in view of the high mortality rate after infection, the research work related to H7N9 avian influenza virus still needs to be carried out in a third-level biosafety laboratory, which is to a large extent Limitations on the development of research related to H7N9 avian influenza

Method used

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  • Preparation method of H7N9 pseudoviruses and applications of H7N9 pseudoviruses
  • Preparation method of H7N9 pseudoviruses and applications of H7N9 pseudoviruses
  • Preparation method of H7N9 pseudoviruses and applications of H7N9 pseudoviruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Construction of pVAX-HA and pVAX-NA vectors

[0041] 1. Obtain the nucleotide sequence of HA (439507) and NA (439509) genes of H7N9 / A / Anhui / 1 / 2013 (EPI_ISL_138739) strain from GISAID;

[0042] 2. Optimize the HA and NA gene sequences according to the principles of mammalian cell codon optimization, and add HindIII and BamHI restriction endonuclease sites at the 5'and 3'of the sequence. The optimized sequence is made by Kings Swiss synthesis; wherein the optimized sequence of the H7N9 hemagglutinin protein gene (HA) mammalian expression codon is shown in SEQ ID NO. 1, and the optimized sequence of the H7N9 neuraminidase gene (NA) mammalian expression codon is shown in SEQ ID NO. 2. Show.

[0043] 3. Double enzyme digestion of pVAX-1 vector with BamHI and HindIII, and recovery of vector fragments;

[0044] 4. Mix the HA and NA fragments with the linearized vector at a molar ratio of 1:5, and ligate with T4 ligase at 16°C for 12 hours

[0045] 5. Transform DH5α competen...

Embodiment 2

[0049] Example 2: pVAX-TMPRSS2 vector construction

[0050] 1. Obtain the nucleotide sequence of TMPRSS2 (BC_051839) gene from NCBI;

[0051] 2. Design primers based on the TMPRSS2 gene sequence, and add a homologous sequence of pVAX-1 vector to the 5'ends of the upstream and downstream primers;

[0052]

[0053] Note: the underlined part is the homologous sequence of the vector

[0054] 3. Extract RNA from Calu-3 cells and reverse transcription to obtain cDNA;

[0055] 4. Amplify TMPRSS2 by PCR, the PCR reaction conditions are: 94°C, 2min; 94°C, 30s, 58°C, 30s, 72°C, 2min, 35 cycles; 72°C, 10min;

[0056] 5. Double digestion of pVAX-1 vector with BamHI and HindIII, and recovery of vector fragments;

[0057] 6. Mix 20ng of PCR product and linearization vector pVAX-160ng, 2×SeamLessMasterMix, add water to make up to 10μL, react and ligate at 50℃ for 15min, and place on ice;

[0058] 7. Take 5μL of connecting solution and add 50μL of just thawed DH5α competent cells, mix gently, and ice bath...

Embodiment 3

[0064] Example 3: H7N9 pseudovirus packaging scheme one

[0065] 1. Inoculate 4×10 in a 6-well plate 5 293T cells, cultured to 80% confluence;

[0066] 2. Mix the plasmid pNL-4.3.luc.ER- and pVAX-HA according to 3:1 (unit: μg), add 64μL solutionB (transfection reagent components, transfection reagents provided by Shenzhen Angran Biotech Co., Ltd.) and mix to obtain solution1 ;

[0067] 3. Mix 128μLsolutionB and 64μLsolutionA (transfection reagent components, transfection reagents provided by Zhenanran Biological Company) to obtain solution2;

[0068] 4. Add solution2 dropwise to solution1, gently blow and mix, and let stand at room temperature for 30 minutes;

[0069] 5. Add the DNA-liposome complex dropwise to the 293T cell culture medium and incubate at 37°C;

[0070] 6. Discard the culture medium after 5 hours, wash the cells once with PBS, and replace with fresh Opti-DMEM culture medium containing 0.5 μg / ml of bacterial neuraminidase;

[0071] 7. Continue to incubate for 48 hours, ha...

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Abstract

The invention discloses a preparation method of H7N9 pseudoviruses and applications of H7N9 pseudoviruses, which belong to the field of bio-medicine techniques. The preparation method comprises the following steps: cloning HA and NA gene sequences of H7N9 avian influenza viruses to mammalian expression vectors pVAX-1; and then, co-transfecting 293T cells by using pVAX-HA and retrovirus vectors pNL-4.3. Luc. E-R-isoplasmids, so that H7N9 pseudoviruses are generated. Cytostatics and neuraminidase inhibitors for inhibiting the entry of H7N9 avian influenza viruses are screened by using H7N9 pseudoviruses and a pseudovirus cell model, studies on influenza virus neutralizing antibodies are performed by using H7N9 pseudoviruses, and influenza virus infections are prevented and treated by using H7N9 pseudoviruses as anti-influenza immunological preparations.

Description

Technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a method for preparing an H7N9 pseudovirus and an application of the H7N9 pseudovirus. Background technique [0002] Influenza has always been one of the main threats to human health. The Spanish flu in 1918, the H1N1 flu epidemic in 2009, and the bird flu A / H5N1 brought great disasters or losses to humans. Since 2013, the A / H7N9 avian influenza outbreak in eastern China has caused more than 45 deaths. Its risks and epidemic potential have attracted worldwide attention. Although H7N9 avian influenza is a low pathogenic avian influenza virus, in view of its high mortality rate after infection, at present, research work related to H7N9 avian influenza virus still needs to be carried out in a third-level biosafety laboratory, which is to a large extent The above limits the development of H7N9 bird flu related research. Summary of the invention [0003] In view of the pr...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C07K16/08C12Q1/02A61K35/22A61P31/16
Inventor 吴艳玲张文申立文
Owner 杭州庆正鸿科技有限公司
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