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Trichina paramyosin gene and application thereof

A Trichinella spiralis, amino acid technology, applied in the fields of application, anti-animal/human immunoglobulin, gene therapy, etc., can solve the problems of complexity and diversity of Trichinella spiralis antigens, limited antigen acquisition, etc.

Inactive Publication Date: 2007-07-18
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because pathogens cannot be subcultured in large quantities in vitro, the acquisition of antigens is limited; coupled with the complexity and diversity of Trichinella spiralis antigens, there is currently no good antigen with high sensitivity and high specificity as a candidate antigen for diagnostic or protective vaccines molecular

Method used

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  • Trichina paramyosin gene and application thereof
  • Trichina paramyosin gene and application thereof
  • Trichina paramyosin gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1 Cloning and sequence analysis of Trichinella spiralis paramyosin gene

[0093] 1. Preparation of screening serum (artificially immunized rabbit serum and artificially infected rabbit serum)

[0094] Adults of Trichinella spiralis Heilongjiang (International Standard Strain Code ISS533) isolated from pigs were collected, and the adult bodies were placed in a grinder and rapidly ground for 15 minutes under ice bath. Transfer the ground liquid into a 10ml centrifuge tube, and ultrasonicate (on ice) 3 times, 3 minutes each time, with a 1-minute rest in between. Repeated freezing and thawing several times, centrifuging and taking the supernatant to obtain the whole worm soluble antigen. Immunize Japanese big-eared white rabbits (1.6mg / rat), boost once a week, and boost 4 times to obtain artificially immune rabbit serum; experimental rabbits were fed with 4000 Trichinella muscle larvae, and artificially infected rabbit serum was obtained after 4 weeks. As determin...

Embodiment 2

[0165] Example 2 Preparation of Paramyosin Gene Recombinant Protein and Artificial Immunization Rat Serum

[0166] 1. Prokaryotic expression and purification of paramyosin gene

[0167] According to the Ts86 cDNA sequence, a pair of primers were designed, the upstream primer was 5'-CGGGATCCATGTCTCTGTATCGCAGTCCCAGT-3' (SEQ ID NO: 3) (32 nt in total, containing a BamH I restriction site), and the downstream primer was 5'-CGGAATTCATATTCATGTCCTTCTCCATCAC-3 '(SEQ ID NO: 4) (total 32nt, containing an EcoR I restriction site). The Ts86 cDNA clone obtained from the immune screening library was used as a template for PCR, and the product was subcloned into the prokaryotic expression vector PET-28a(+) (Novagen Company) after being digested with BamH I and EcoR I. The correct recombinant plasmid was transformed into Escherichia coli BL21 strain (Novagen Company), and induced by 1.0mM IPTG at 150 rpm at 37°C for 3 hours to obtain the recombinant protein of the paramyosin gene (885 amino ...

Embodiment 3

[0169] Embodiment 3 Immune characteristics of paramyosin gene recombinant protein

[0170] 1. Enzyme-linked immunosorbent assay (ELISA) detection

[0171] 1 μg of paramyosin gene recombinant protein (prepared in Example 2) was used as an antigen to coat a 96-well microtiter plate, the primary antibody was the serum to be tested, and the secondary antibody was the corresponding horseradish peroxidase-labeled (goat anti-human, goat Anti-rabbit or goat anti-pig) secondary antibody, normal human serum, normal rabbit serum, and normal pig serum were used as negative controls. According to the OD value of the serum sample to be tested is greater than 2.1 times of the negative control, it is a positive judgment result. After ELISA detection, the positive rate of trichinellosis patient serum (23 copies) was 86.96%, trichinellosis rabbit serum (2 copies) and trichinellosis pig serum (4 copies) were all positive, indicating that the recombination of the paramyosin gene The protein has...

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Abstract

The present invention relates to paramyosin gene of new trichina antigen, constitution of the vector containing the gene, prokaryon expression vector containing the gene and the expressed protein. The present invention also relates to the application of the gene and its coded protein. When the recombinant protein of trichina paramyosin gene is used in immunizing animal, high titer antibody and effective immunoprotection against attacting infection will generate.

Description

field of invention [0001] The invention relates to a trichinella spiralis new antigen gene paramyosin gene, the construction of a vector containing the gene, the prokaryotic expression vector, the expressed protein and the antibody produced by the protein-immunized animal. The present invention also relates to the application of said gene and protein. Background technique [0002] Trichinella spiralis (Trichinella spiralis, referred to as Trichinella spiralis), can infect humans and more than 150 kinds of animals. Huge economic losses to animal husbandry. The life history of Trichinella spiralis is briefly described as follows: When a human or animal host eats raw or semi-raw meat (pork, dog meat, etc.) containing Trichinella spiralis cysts, the larvae in the cysts escape under the action of gastric juice and intestinal juice and develop into adults in the small intestine. After mating, the females produce new larvae. The newborn larva invades the lymphatic vessels or vei...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/30C07K14/44C12P21/02C07K16/18A61K38/16C12Q1/68G01N33/53G01N33/68A61K31/7088A61K48/00C07K19/00
Inventor 诸欣平杨静杨雅平顾园李强
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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