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Specific PPMP-type antigen of Clonorchis sinensis

A technology for Clonorchis sinensis and Clonorchis sinensis, which is applied in the directions of antibodies, anti-infective drugs, and antibody medical components, and can solve the problems of low specificity and sensitivity.

Active Publication Date: 2014-07-02
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

[0013] The purpose of the present invention is to provide three kinds of specific antigens of Clonorchis sinensis and applications thereof, and the specific antigens and applications thereof of these three kinds of Clonorchis sinensis will solve the prior art method for diagnosing Clonorchis sinensis Technical issues with low specificity and sensitivity

Method used

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  • Specific PPMP-type antigen of Clonorchis sinensis
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  • Specific PPMP-type antigen of Clonorchis sinensis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1 Construction of Clonorchis sinensis adult cDNA library

[0076] 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, and then dissect them, collect the adults of Clonorchis sinensis, wash them with sterilized saline, and store them in liquid nitrogen.

[0077] TRIzol (GIBCO / BRL company) kit was used to extract the total RNA of Clonorchis sinensis adults (1 gram of Clonorchis sinensis adult worms, stored in liquid nitrogen).

[0078] The mRNA was purified from the extracted total RNA using the mRNA Purification Kit (mRNA Purification Kit, Amersham Company). See the product manual for specific steps.

[0079] Clonorchis sinensis adult cDNA library was constructed by directional cloning method using ZAP-cDNA Synthesis Kit (Stratagene Company). Follow the instructions of the kit to operate, the main process includes:

[0080] 1. Fo...

Embodiment 2

[0090] Example 2 Immunological screening of cDNA library

[0091] Phage infection:

[0092] Take 200 μl of XL1-blue MRF′ bacteria solution and mix it with an appropriate amount of cDNA library phage solution (predetermined according to the library titer, about 3000 phage plaques / plate), incubate at 37°C for 15 minutes, then add 3ml of 48°C upper layer agar ( top agar) and spread on NZY medium plates immediately. Solidify at room temperature for 10 minutes and incubate at 42°C.

[0093] Inducible expression of fusion proteins:

[0094] 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 blockin...

Embodiment 3

[0099] Example 3 Deletion and circularization of phage into pBluescript SK_phagemid and extraction of phagemid

[0100] E.coli XL1-blue MRF' was cultured overnight in LB medium (containing 10mM MgSO4, 0.2% maltose, 15μg / 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 relates to a specific PPMP-type antigen of Clonorchis sinensis. The nucleotide sequences of the antigen gene are shown in SEQIDNO: 3. The invention also provides an antibody specifically bound to the specific antigen of Clonorchis sinensis, an isolated specific PPMP-type antigen protein of Clonorchis sinensis encoded by the nucleotide sequences of the specific antigen genes of Clonorchis sinensis, a vector containing the specific antigen genes of Clonorchis sinensis and a kit. The invention also provides an application of the specific antigen protein of Clonorchis sinensis, the antibody, vectors and the kit in the preparation of a medicament for the treatment, diagnosis or prevention of clonorchiasis. The specific PPMP-type antigen of Clonorchis sinensis, which is disclosed by the invention, has a higher immunogenicity, and easily used for preparing the multi / monoclonal antibody and can be applied to antigen detection and other aspects. Experimental results show that the experimental animals (mice) have a stronger immune response to the antigen.

Description

[0001] This application is a divisional application with application number "2010101786846", application date "May 19, 2010", and invention title "Clonorchis sinensis-specific PPMP-type antigen and its application". Technical field: [0002] The invention belongs to the field of bioengineering, and in particular relates to a clonorchis sinensis, specifically three specific antigens of clonorchis sinensis PPMP-type antigens and applications thereof. Background technique: [0003] Clonorchiasis sinensis (Clonorchiasis sinensis) is a zoonosis caused by the infection of 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 ha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C07K16/18C12N15/12C12N15/63A61K39/00A61K39/395A61P33/10G01N33/68
CPCA61K39/00A61K2039/505C07K14/43559C07K16/18G01N33/6803G01N2333/43547
Inventor 许学年周岩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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