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Trichinella Spiralis larva ES antigen gene vaccine and preparation method

An antigen gene and muscle larvae technology, applied in the field of Trichinella muscle larvae ES antigen gene vaccine and preparation thereof, can solve the problems of complex components of the Trichinella muscle larva ES antigen composition, and achieve easy reversal of elimination and restoration of original activity and structure. simple effect

Active Publication Date: 2013-10-16
JILIN AGRICULTURAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ES antigens are composed of a variety of protein components. Despommier et al. used SDS-PAGE to find 28 antigenic components in Trichinella spiralis ES antigens. Using IEF method, 37 antigenic components could be found, including 22 glycoproteins; domestic scholars such as Zhu Xingquan also The composition of the ES antigen of Trichinella spiralis muscle larvae is very complex through experimental research and analysis

Method used

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  • Trichinella Spiralis larva ES antigen gene vaccine and preparation method
  • Trichinella Spiralis larva ES antigen gene vaccine and preparation method
  • Trichinella Spiralis larva ES antigen gene vaccine and preparation method

Examples

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Embodiment 1

[0033] Example 1 Acquisition of 43ku and 45ku antigen genes of Trichinella spiralis muscle larvae

[0034] (1) Primer design and synthesis

[0035] According to the gene sequences of the 43ku and 45ku ES antigens of Trichinella spiralis muscle larvae registered in GenBank, primers were designed using Primers 5.0, and the upstream and downstream primers of the target gene were designed respectively Kpn I and Bam H Ⅰ Restriction site, the primer sequence was synthesized by Shanghai Sangon Bioengineering Co., Ltd. Primer sequences are as follows

[0036] 43ku upstream primer (43-P1): 5'-CGC GGTACC ATGCGAATATACATTTTTCTTAG-3'

[0037]43ku downstream primer (43-P2): 5'-CGA GGATCC TTAGCTGTATGGGCAA-3'

[0038] 45ku upstream primer (45-P1): 5'-CGC GGTACC ATGAAACTCTTGCTTTTAACA-3'

[0039] 45ku downstream primer (45-P2): 5'-GC GGATCC TTAGCCTTGCTTAGAGAG-3'

[0040] (2) PCR amplification of 43ku and 45ku antigen genes

[0041] Trichinella spiralis muscle larvae were prep...

Embodiment 2

[0054] Example 2 Eukaryotic recombinant expression vector pVAXⅠ- 43ku and pVAXⅠ- 45ku preparation of

[0055] The eukaryotic expression vector pVAXⅠ was purchased from Invitrogen Corporation of the United States, and pVAXⅠ was prepared according to the instructions of the plasmid mini-extraction kit, such as image 3 shown.

[0056] (1) Recovery, ligation and transformation of the target gene

[0057] use Kpn Ⅰ and restriction endonuclease respectively for pMD8T- 43ku , pVAXⅠ-45ku and pVAXⅠ were subjected to double enzyme digestion to obtain 43ku, 45ku ES antigen gene and pVAXⅠ large fragment; the double enzyme digestion system is as follows:

[0058]

[0059] Reaction conditions: 37°C water bath for 3 h. After the end, follow the instructions of the DNA gel recovery kit to recover the target gene fragment.

[0060] The 43ku and 45ku ES antigen genes were respectively connected to the large fragment of pVAXⅠ vector. The connection system is as follows:

[0061...

Embodiment 3

[0067] Example 3 pVAXⅠ- 43ku and pVAXⅠ- 45ku Transfection of MA104 cells and identification of expression products

[0068] Follow Lipofectamine TM 2000 instruction manual, pVAXⅠ- 43ku , pVAXⅠ- 45ku MA-104 cells were transfected, and pVAXⅠ was used as blank control.

[0069] After the transfection, the MA-104 cells were covered with coverslips and taken out, dried and washed once with PBS (pH7.2), fixed with 10% acetone for 10-15 min, washed three times with PBS (pH7.2), After blotted the edge moisture, add mouse-derived 43ku and 45ku ES antigen monoclonal antibodies respectively, place in a 37°C incubator for 2 hours, take out the cover slip and wash it with PBS (pH7.2) for 3 times, add FITC-labeled goat anti-mouse IgG Antibody, placed in a 37°C incubator for 2 hours, took out the cover slip and washed it with PBS (pH7.2) for 3 times, blotted the edge of the water and stained it with Evans blue, observed under a fluorescent microscope, MA after transfection of the r...

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Abstract

The invention discloses a Trichinella Spiralis larva ES antigen gene vaccine and a preparation method thereof. Trichinella Spiralis larva 43ku and 45ku ES protein genes are respectively inserted to a pVAXI eukaryotic expression vector to obtain recombinant plasmids, and the recombinant plasmids are mixed according to a ratio of 1:1 to prepare a combined recombined DNA vaccine for preventing Trichinella Spiralis infection and improving the worm reduction rate of bodies. After the injection immunization of hindlimb muscles of mice for three weeks, 2000 worms are killed, the worm reduction rate can reach 76.65%, and the quantity of eggs at the tongue tissues of the mice is obviously reduced.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to a trichinella spiralis muscle larva ES antigen gene vaccine and a preparation method thereof. Background technique [0002] Trichinella spiralis parasitizes humans and various vertebrates, and is a zoonotic parasitic disease that causes trichinellosis. Trichinella can parasitize humans and more than 150 kinds of animals. The main cause of infection is the intake of raw or undercooked meat products with cysts of Trichinella muscle larvae. resulting in the death of the patient. It is estimated that there are approximately 11 million infected people in the world at present. After 1975 in my country, there were also reports of outbreaks of this disease in Jilin, Liaoning, Heilongjiang, Henan, Hubei and other provinces and municipalities. The disease not only seriously endangers human health, but also causes huge economic losses to the pig industry and meat export. The Internation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/79C12N15/12A61K48/00A61P33/00
Inventor 杨桂连王春凤杨文涛刘高升赵葛
Owner JILIN AGRICULTURAL UNIV
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