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

A technology of swine influenza virus and porcine pox virus, applied in botany equipment and methods, biochemical equipment and methods, antiviral agents, etc., can solve the problem that heterologous and heterogeneous viruses cannot provide effective protection, and it is difficult to effectively control swine influenza virus Infection and transmission, weak ability of cellular immunity, etc., to achieve the effects of screening and purification, low cost, and broad application prospects

Inactive Publication Date: 2012-10-17
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 swine pox virus transfer vector pUSZ11 / H1

[0049] 1.1 Construction of poxvirus vector pUSZ11

[0050] The map of pUSZ11 plasmid (constructed by Huang Dongyan) is as follows figure 2 As shown, the full length is 8391bp; LF in the pUSZ11 plasmid is the homologous recombination arm of hogpox virus on the left, which is homologous to 12121-13268bp of the Kasza strain of hogpox virus. With wild-type swine pox virus wtSPV (purchased from ATCC, the deposit number is number VR-363 TM , the same below) genomic DNA as a template, PCR is carried out with a pair of primers SEQ ID NO.2 and SEQ ID NO.3, then the amplified product is 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 homologous recombination arm of the right hogpox virus, which is homologous to the 13457-14831 bp of the hogpox virus Kasza strain. Also use wtSPV genomic DNA as a template, use primers SEQ ID NO.5 and S...

Embodiment 2

[0062] Example 2 Construction and Identification of Swine Influenza Virus H1N1 Subtype HA1 Protein Recombinant Pox Virus

[0063] 2.1 Infection and transfection

[0064] In a 6-well plate, culture PK15 cells (purchased from ATCC, Number: CCL-33 TM , the same below) to form a single layer, discard the nutrient solution; infect with 0.02 MOI of wild-type swine pox virus wtSPV, act at 37°C for 2 hours, shake 3 times during this time, discard the infection solution, wash once with the lotion, add 2 mL to each well Antibiotic-free maintenance solution. Take 10 μL of liposome (product of Invitrogen) and add it to 250 μL of serum-free MEM, mix gently, and let it stand 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, place them at room temperature for 20 min, add the complex to a 6-well plate, and shake back and forth to mix well. After 6 hours at 37°C, the transfection solution was discar...

Embodiment 3

[0077] The immunological test of embodiment 3 mice

[0078] Forty-five female BALB / c mice aged 6-8 weeks were randomly divided into 3 groups, 15 in each group. Each muscle of the first group was immunized with rSPV / H1 (CCTCC NO: V201101) at a dose of 0.2×10 7. 0TCID 50 ; The second group was intramuscularly injected with wtSPV 0.2×10 7.0 TCID 50 , each group 3 was intramuscularly injected with 0.2 mL of PK15 cell lysed supernatant. After 21 days and 35 days, the same dose was used to boost the immunization twice. Blood samples were taken at 21, 35, and 42 days after the first immunization to determine neutralizing antibodies; spleens were collected at 21, 35, and 42 days after the first immunization, lymphocytes were separated, and lymphocyte proliferation responses were measured (5 mice / group / time); 35 days after the first immunization Take spleen, separate lymphocytes, stimulate with SIVH1N1 (A / swine / Shanghai / 1 / 2005 (H1N1)) (separated by Dr. Qi Xian. GenBank accession n...

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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 carrier, a large number of foreign genes can be accepted and 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, which can stimulate the host to produce an effective immune response. Hogpox virus does not infect other animals, but only infects pigs, and produces only mild reactions in pigs, so it is relatively safe to use. Therefore, hogpox virus is an ideal carrier for delivering immunogens to pigs. The recombinant swine pox virus vaccine has the antigen multiplication ability of the attenuated vaccine, ...

Claims

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Application Information

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Patent Type & Authority Patents(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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