Construction method of SAG2 gene, ROP9 gene and MIC3 gene recombinant adenovirus, recombinant adenovirus and application

A technology of recombinant adenovirus and gene recombination, which is applied in the field of genetic bioengineering, can solve the problems of lack of Toxoplasma gondii vaccine and achieve effective immune protection

Active Publication Date: 2019-04-02
SHENYANG AGRI UNIV
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Problems solved by technology

However, there is no Toxoplasma vaccine available for humans, and there is only one veterinary Toxoplasma vaccine used in some countries: S48 attenuated strain vaccine

Method used

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  • Construction method of SAG2 gene, ROP9 gene and MIC3 gene recombinant adenovirus, recombinant adenovirus and application
  • Construction method of SAG2 gene, ROP9 gene and MIC3 gene recombinant adenovirus, recombinant adenovirus and application
  • Construction method of SAG2 gene, ROP9 gene and MIC3 gene recombinant adenovirus, recombinant adenovirus and application

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Embodiment Construction

[0027] Below in conjunction with accompanying drawing and specific embodiment the present invention is described in further detail:

[0028] Compared with other vectors, adenovirus vectors have obvious advantages. There are many reports at home and abroad on the preparation of vaccines using adenoviruses as vectors. At present, the adenovirus vector system is quite mature. The research on T.gondii is also quite clear. It has been reported that other vectors are used to develop the genetically engineered vaccine of SAG2, ROP9 or MIC3, and the immune challenge test proves that mice have a certain protective effect. Therefore, the present invention utilizes T.gondii SAG2, ROP9 and MIC3 genes were used to construct a recombinant adenovirus Ad-SAG2-ROP9-MIC3-EGFP, so as to develop a trivalent genetically engineered live vector vaccine for preventing toxoplasmosis.

[0029] T.gondii SAG2 gene of the present invention, ROP9 gene and MIC3 gene construct the method for recombinant aden...

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Abstract

The invention discloses a construction method of an SAG2 (surface antigen 2) gene, ROP9 (rhoptry protein 9) gene and MIC3 (microneme protein) gene recombinant adenovirus, a recombinant adenovirus andan application. The construction method comprises the following steps of amplifying an SAG2 gene, an ROP9 gene and a MIC3 gene from a Toxoplasma gondii RH strain, respectively; cloning the obtained target genes into a shuttle vector pHBAd-EF1a-MCS-3flag-CMV-EGFP; co-transfecting HEK293A cells with the recombinant adenovirus shuttle plasmid pHBAd-SAG2-ROP9-MIC3-EGFP and an adenovirus large skeletonpBHGlox(delta)E1, 3Cre by a liposome transfection method; and performing recombining and packaging to obtain the recombinant adenovirus Ad-SAG2-ROP9-MIC3-EGFP containing the SAG2 gene, the ROP9 geneand the MIC3 gene. After the recombinant adenovirus disclosed by the invention is used for immunizing Balb/c mice, the antibody level of the mice is notably increased, the proportion of activated TCLand Th lymph cells is notably increased, cytokines of IF-gamma, IL-6, TN-alpha and the like are notably increased, and the recombinant adenovirus produces definite protective effects on the Balb/c mice.

Description

technical field [0001] The invention belongs to the field of genetic bioengineering and relates to a method for constructing a recombinant adenovirus, specifically, a method for constructing a recombinant adenovirus using Toxoplasma gondii SAG2 gene, ROP9 gene and MIC3 gene, and the recombinant adenovirus and its application. Background technique [0002] Toxoplasma gondii (Toxoplasma gondii) is an obligate intracellular nucleated parasitic protozoan. It is a serious food-borne zoonotic parasite that can infect more than 200 species of animals including livestock, poultry and humans. The human infection rate is about 25% to 50%, and there are about 500 to 1 billion people infected with toxoplasmosis in the world; the lethal outbreak of toxoplasma can cause a large number of livestock and poultry deaths, and the death rate of pigs can reach 60%. Toxoplasma gondii not only directly endangers the development of animal husbandry and product quality and safety, but also can cause...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N7/01A61K39/002A61P33/02
CPCA61K39/002A61P33/02C12N7/00C12N15/86A61K2039/5256A61K2039/552C12N2710/10352C12N2710/10343C12N2800/107
Inventor 陈启军张东超姜宁
Owner SHENYANG AGRI UNIV
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