Specific antigen of clonorchis sinensis and application thereof

A technology for Clonorchis sinensis and Clonorchis sinensis, which is applied to invertebrate antigen components, medical preparations containing active ingredients, and applications, etc., can solve the problems of low specificity and sensitivity, and can prevent Effects of orchidism infection

Active Publication Date: 2012-05-23
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 specific antigen of Clonorchis sinensis and its use, which will solve the technical problems of low specificity and sensitivity in the diagnosis of Clonorchis sinensis in the prior art.

Method used

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  • Specific antigen of clonorchis sinensis and application thereof
  • Specific antigen of clonorchis sinensis and application thereof
  • Specific antigen of clonorchis sinensis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of Clonorchis sinensis adult cDNA library

[0047] Five cats were infected with Clonorchis sinensis metacercariae collected from Guangxi Zhuang Autonomous Region by intragastric administration. After 40 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.

[0048] 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.

[0049] 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.

[0050] 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:

[0051] 1. For the ...

Embodiment 2

[0061] Example 2 Immunological screening of cDNA library

[0062] Phage infection:

[0063] 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.

[0064] Inducible expression of fusion proteins:

[0065] 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 ...

Embodiment 3

[0070] Example 3 Deletion and circularization of phage into pBluescript SK_phagemid and extraction of phagemid

[0071] E. coli XL1-blue MRF' was cultured overnight in LB medium (containing 10 mM MgSO4, 0.2% maltose, 15 μg / ml Tet). On the next day, 100 μl of the culture solution was transferred to a new LB medium, and cultured at 37° C. with shaking at 200 rpm for about 2 hours. The culture solution was centrifuged at 2000g for 10 minutes to pellet the bacteria and resuspended to OD with 10mM MgSO4 600 = 1.0. Add 200 μl of E.coli XL1-blue MRF' suspension, 50 μl of SM buffer containing positive phages and 1 μl of helper phages to the bacterial culture tube. Place the bacterial culture tube in a 37°C water bath for 15 minutes. Then 3 ml of LB was added, and cultured with shaking at 37° C. for 5 hours. Take out the bacterial culture tube, heat at 65°C for 20 minutes, then centrifuge at 3000g for 15 minutes, transfer the supernatant to a new centrifuge tube, and store at 4°C. ...

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Abstract

The invention provides a specific antigen of clonorchis sinensis, and also provides a specific antibody combining the specific antigen of the clonorchis sinensis, a separated protein which is coded by a nucleotide sequence of a specific antigenic gene of the clonorchis sinensis, a vector containing the specific antigenic gene of the clonorchis sinensis, a vaccine, a kit and application of the specific antigen of the clonorchis sinensis, the antibody, the vaccine and the kit in preparing a drug for treating, diagnosing or preventing the clonorchiosis. The Cs1 antibody 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 belongs to the field of bioengineering, and in particular relates to a clonorchis sinensis, specifically a specific antigen of the clonorchis sinensis and its application. Background technique: [0002] Clonorchiasis sinensis is a zoonotic disease caused by Clonorchis sinensis [Clonorchis sinensis, Cs]. 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 a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/435C07K16/18C12N15/63C12N1/21C12N5/10A61K39/00A61K39/395A61K48/00A61P33/10G01N33/53
CPCA61K39/0003A61K38/00C07K14/43559A61P33/10
Inventor 许学年冯正董玉婷周岩包意芳徐斌
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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