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Specific GRA2a (Glycine Rich Antigen)-type antigenic protein of clonorchis sinensis and application thereof

A technology for Clonorchis sinensis and Clonorchis sinensis, which is applied in the direction of anti-animal/human immunoglobulins, applications, antibodies, etc., and can solve the problems of low specificity and sensitivity of Clonorchis sinensis.

Active Publication Date: 2013-11-13
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] The object of the present invention is to provide a kind of Clonorchis sinensis-specific GRA2a class antigen protein, described this Clonorchis sinensis-specific GRA2a class antigen protein will solve the specificity and the specificity of diagnosing Clonorchis sinensis disease in the prior art Less sensitive technical issues

Method used

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  • Specific GRA2a (Glycine Rich Antigen)-type antigenic protein of clonorchis sinensis and application thereof
  • Specific GRA2a (Glycine Rich Antigen)-type antigenic protein of clonorchis sinensis and application thereof
  • Specific GRA2a (Glycine Rich Antigen)-type antigenic protein of clonorchis sinensis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Construction of Clonorchis sinensis adult cDNA library

[0042] Five cats were infected with Clonorchis sinensis metacercariae collected from Guangxi Zhuang Autonomous Region by intragastric administration. After 42 days, check the feces for eggs, then dissect them, collect the adults of Clonorchis sinensis, wash them with sterilized saline, and store them in liquid nitrogen.

[0043] The total RNA of Clonorchis sinensis adults (1 g of Clonorchis sinensis adult worms packed, stored in liquid nitrogen) was extracted using TRIzol (GIBCO / BRL company) kit.

[0044] mRNA was purified from the extracted total RNA using an mRNA Purification Kit (mRNA Purification Kit, Amersham Company). See the product manual for specific steps.

[0045] Clonorchis sinensis adult cDNA library was constructed by directional cloning method using ZAP-cDNASynthesis Kit (Stratagene Company). Follow the instructions of the kit to operate, the main process includes:

[0046] 1. For the ...

Embodiment 2

[0056] Example 2 Preparation of Clonorchis sinensis ESA-immunized mouse serum

[0057] The obtained fresh Clonorchis sinensis adults were washed several times with sterile physiological saline, transferred to Tyrode's medium (containing 10 μg / ml each of diabodies and amphotericin B), cultured in 5% CO2 at 37°C. The adult culture is based on centrifugation at 3000g for 15 minutes to remove the precipitate (worm eggs). The supernatant was then ultracentrifuged at 20,000 g to remove sediments that may contain worm fragments. The resulting supernatant is excreted secreted antigen (ESA).

[0058] Sera from immunized mice were prepared routinely. Balb / c mice were inoculated with 30 μg of antigen, and used Freund's complete adjuvant (Sigma Company) for the first inoculation, and injected subcutaneously; every 3 weeks, they were boosted with Freund's incomplete adjuvant (Sigma Company), intraperitoneally injected, and boosted immunization for 4 days in total. Second-rate. The bloo...

Embodiment 3c

[0059] The immune screening of embodiment 3cDNA library

[0060] Phage infection:

[0061] Take 200 μl of XL1-blue MRF′ bacteria solution and mix with an appropriate amount of cDNA library phage solution (predetermined according to the library titer, about 3000 plaques / per plate), incubate at 37°C for 15 minutes, add 3ml of 48°C upper agar ( top agar) and spread on NZY medium plates immediately. Solidify at room temperature for 10 minutes and incubate at 42°C.

[0062] Inducible expression of fusion proteins:

[0063] After observing the appearance of phage plaques (about 3.5 hours), cover the plate with a nitrocellulose membrane (Hybond-C extra, Amersham, NC) soaked in IPTG (15mM / L) for 30 minutes, and incubate at 37°C for another 3.5 hours; Take out the plate and cool it at 4°C for 15 minutes, then mark the film and the plate with a needle. Remove the membrane and wash it in TBST for 3 times, each time for 10 minutes, then soak the NC membrane in blocking solution, shake...

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Abstract

The invention provides a specific GRA2a (Glycine Rich Antigen)-type antigenic protein of clonorchis sinensis, and also provides a specific antibody combining the specific GRA2a-type antigenic protein of the clonorchis sinensis, an antigenic gene used for coding the specific GRA2a-type antigenic protein of the clonorchis sinensis, a vector containing the specific GRA2a-type antigenic gene of the clonorchis sinensis, a kit and application of the specific GRA2a-type antigenic protein of the clonorchis sinensis, the antibody and the kit in preparing a drug for preventing, treating and diagnosing the clonorchiosis. The GRA2a-type antigen of the clonorchis sinensis has higher immunogenicity, can be used for easily preparing a polyclonal / monoclonal antibody, and is further applied to the aspects such as antigen detection and the like. Moreover, the stronger immune response of an experimental animal (mouse) on the antigen is realized.

Description

Technical field: [0001] The invention relates to the field of bioengineering, in particular to Clonorchis sinensis, especially a Clonorchis sinensis-specific GRA2a antigen and application thereof. Background technique: [0002] Clonorchiasis sinensis is a zoonotic disease caused by Clonorchis sinensis [Clonorchis sinensis, C.s]. The disease is mainly distributed in Asia, such as China, Japan, South Korea (the main human parasite in South Korea), North Korea, Vietnam, Southeast Asia and other countries. An estimated 35 million people are infected worldwide. In my country, except Xinjiang, Inner Mongolia, Gansu, Qinghai, Tibet, Ningxia and other provinces and autonomous regions, no reports have been reported, the remaining 25 provinces, municipalities, autonomous regions, Taiwan Province and Hong Kong Special Administrative Region have reported the prevalence or case reports of the disease . Case reports of the disease are also increasing in non-endemic regions and developed...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C07K16/18C12N15/12C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A61K39/00A61K39/395A61K48/00A61P33/12G01N33/577
Inventor 许学年周岩卢艳董玉婷包意芳徐斌冯正
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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