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Swine influenza virus H1N1 subtype hemagglutinin (HA)-1 protein recombinant suipoxvirus and preparation method thereof

A swine influenza virus, H1N1 technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, antiviral agents, etc., can solve the problem of difficult to effectively control the infection and spread of swine influenza virus, weak cellular immunity, heterologous and heterotypic viruses can not provide effective protection and other problems, to achieve the effect of facilitating screening and purification, low cost, and convenient use

Inactive Publication Date: 2011-08-17
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the ability of inactivated vaccines to induce cellular immunity is weak, and they cannot provide effective protection against heterologous and heterogeneous viruses, and it is difficult to effectively control the infection and spread of swine influenza viruses with variable antigens in pigs
At present, there is no report on the construction of swine influenza virus H1N1 subtype HA1 protein recombinant swine pox virus

Method used

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  • Swine influenza virus H1N1 subtype hemagglutinin (HA)-1 protein recombinant suipoxvirus and preparation method thereof
  • Swine influenza virus H1N1 subtype hemagglutinin (HA)-1 protein recombinant suipoxvirus and preparation method thereof
  • Swine influenza virus H1N1 subtype hemagglutinin (HA)-1 protein recombinant suipoxvirus and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Construction of swinepox virus transfer vector pUSZ11 / H1

[0049] 1.1 Construction of swine pox virus vector pUSZ11

[0050] The map of pUSZ11 plasmid (constructed by Huang Dongyan) is as follows figure 2 As shown, the total length is 8391 bp; the LF in the pUSZ11 plasmid is the left swine pox virus homologous recombination arm, which is homologous to the 12121-13268 bp of swine pox virus Kasza strain. The wild-type swine pox virus wtSPV (purchased from ATCC, deposit number is number VR-363 TM , The same below) genomic DNA was used as a template, a pair of primers SEQ ID NO. 2 and SEQ ID NO. 3 were used for PCR, and then the amplified product was digested with EcoR I and Kpn I, and LF (SEQ ID NO. 4, 1148bp ) Was cloned into pUC19; the RF in the pUSZ11 plasmid is the swine pox virus homologous recombination arm on the right, which is homologous to the 13457-14831 bp of swine pox virus Kasza strain. Similarly, using wtSPV genomic DNA as a template, PCR was perfor...

Embodiment 2

[0062] Example 2 Construction and identification of swine influenza virus H1N1 subtype HA1 protein recombinant swine pox virus

[0063] 2.1 Infection and transfection

[0064] Culture PK15 cells (purchased from ATCC, with antibiotic-free MEM (purchased from TIANGEN BIOTECH, the same below) nutrient solution in a 6-well plate Number: CCL-33 TM , The same below) until a monolayer is formed, discard the nutrient solution; infect with wild-type swine pox virus wtSPV of 0.02 MOI, act for 2 hours at 37°C, shake 3 times during this period, discard the infection solution, wash with lotion once, add 2 mL to each well No antibiotic maintenance solution. Take 10 μL of liposomes (product of Invitrogen) and add it to 250 μL of serum-free MEM, mix gently, and leave it at room temperature for 5 minutes; add 4 μg of the identified plasmid to 250 μL of serum-free MEM and mix gently. Mix the two tubes evenly, let them act at room temperature for 20 minutes, add the complex to the 6-well plate, sh...

Embodiment 3

[0077] Example 3 Immunological test in mice

[0078] Take 45 female BALB / c mice aged 6-8 weeks and randomly divide them into 3 groups with 15 mice in each group. Each muscle of group 1 is immune to rSPV / H1 (CCTCC NO: V201101), the dose is 0.2×10 7. 0TCID 50 ; In the second group, each animal was injected with wtSPV 0.2×10 intramuscularly 7.0 TCID 50 In the third group, 0.2 mL of PK15 cell lysis supernatant was injected intramuscularly. After 21d and 35d, they were boosted twice with the same dose. Blood was collected at 21, 35, and 42 days after the first immunization to determine neutralizing antibodies; at 21, 35, and 42 days after the first immunization, the spleen was taken, lymphocytes were separated, and lymphocyte proliferation was measured (5 mice / group / time); 35 days after the first immunization The spleen was taken, lymphocytes were separated, and stimulated with SIVH1N1 (A / swine / Shanghai / 1 / 2005(H1N1)) (isolated by Dr. Qi Xian. GenBank accession number EU502884), and t...

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Abstract

The invention belongs to the technical field of biology and discloses swine influenza virus H1N1 subtype HA-1 protein recombinant suipoxvirus and a preparation method thereof. An HA1 gene is designed and synthesized according to the gene sequence of HA1 of an A / swine / Shanghai / 1 / 2005(H1N 1) strain of the swine influenza virus, and the gene is cloned in a suipoxvirus vector pUSZ11 to obtain a transfer vector pUSZ11 / H1. The recombinant virus is a positive clone obtained by infecting and transfecting PK15 cells with the suipoxvirus and the transfer vector pUSZ11 / H1 and performing homologous recombination. The recombinant suipoxvirus can express HA1 protein stably, and after infection with the virus, the cytopathy becomes regular, the toxic effect of the breeding virus is stabilized at 107TCID50 / mL, the breeding virus can effectively stimulate the immune protect reaction of organisms and can be used in the preparation of medicines for preventing and / or treating infection with swine influenza virus H1N1 subtype.

Description

Technical field [0001] The invention belongs to the field of biotechnology and relates to a swine influenza virus H1N1 subtype HA1 protein recombinant swine pox virus and a preparation method thereof. Background technique [0002] The poxvirus genome structure is huge, and the large genome provides space for the insertion of many foreign genes. As a vector, it can accept a large number of foreign genes and make them effectively expressed (Barbara E.Straw, Jeffery J. Zimmerman, Sylvie D'Allaire, David J. Taylor.. DISEASES OF SWINE. 9 TH EDITION.[M]). Poxvirus also has good immunogenicity and can stimulate the host to produce an effective immune response. Swine pox virus does not infect other animals, but only infects pigs. It only produces a mild reaction in the pig body and is relatively safe to use. Therefore, swine pox virus is an ideal carrier for delivering immunogen to pigs. The recombinant swine pox virus vaccine has the antigen proliferation ability of an attenuated va...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/863C12N15/44C12N7/01C12N7/02G01N33/569A61K39/145A61P31/16C12R1/93
Inventor 许家荣陆承平黄冬艳
Owner NANJING AGRICULTURAL UNIVERSITY
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