Cryptosporidium parvum recombinant antigen for diagnosis

A technology of Cryptosporidium parvum and recombinant antigen, applied in the field of biological diagnosis

Inactive Publication Date: 2014-04-23
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There is no standardized recombinant antigen diagnostic kit in China, so it is urgent to find new sensitive and spe...

Method used

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  • Cryptosporidium parvum recombinant antigen for diagnosis
  • Cryptosporidium parvum recombinant antigen for diagnosis
  • Cryptosporidium parvum recombinant antigen for diagnosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Cloning of the MUCIN of the Cryptosporidium parvum antigen gene of embodiment 1

[0018] 1. Extraction of Cryptosporidium parvum C. parvum (Changchun isolate) total DNA

[0019] (1) 1×10 7 Add 1ml lysate ((0.5% SDS, 1mM EDTA, 50mM Tris-HCl (pH 8.5)) to two Cryptosporidium parvum to suspend the pellet twice, 14000×g for 5min each time, discard the supernatant;

[0020] (2) Add 100 μL lysate to suspend the precipitate and mix on a vortex shaker for 30 seconds;

[0021] (3) Put it in a 65°C water bath for 1 minute after standing in liquid nitrogen for 1 minute. Repeat this step 18 times, and mix on a vortex shaker for 30 seconds every time you repeat three times;

[0022] (4) Add 1 μL of 20mg / ml proteinase K and let it stand in a water bath at 55°C for 3 hours;

[0023] (5) Stand in a 90°C water bath for 20 minutes to inactivate proteinase K;

[0024] (6) Add 400ul lysate, then add 500μL Tris saturated phenol, shake up and down for 30s, then centrifuge at 12000×g...

Embodiment 2

[0044] Example 2 Construction and expression of recombinant plasmid pMD18-T-MUCIN

[0045] The recombinant plasmid pMD18-T-MUCIN and the expression vector pET-28-a were digested with BamHI and XhoI endonucleases, and the MUCIN gene fragment was recovered by 1% agarose gel electrophoresis and inserted into the expression vector pET-28a to construct the recombinant Expression plasmid pET-28a-MUCIN. For the results of enzyme digestion identification of recombinant expression vectors, see image 3 . The recombinant expression plasmid pET-28a-MUCIN was transferred into BL21(DE3) for expression. The fusion protein was named rMUCIN. The recombinant protein was induced to express in E. coli and its solubility was analyzed. The QIAexpressionist? manual purified 6×His tagged protein for operation .

[0046] Identification of recombinant protein by SDS-PAGE and Western blot: preparation of 12% polyacrylamide gel, Western blot routine method. Primary antibody: mouse anti-HIS tag mon...

Embodiment 3

[0047] Example 3 Using rMUCIN as the detection antigen ELISA to detect anti-Cryptosporidium parvum IgG antibody

[0048] Prepare Cryptosporidium parvum Cp23 antigen according to Priest et al. for serological detection of control antigen (Priest J, Kwon J, Moss D. Detection by enzyme immunoassay of serum immunoglobulin G antibodies that recognize specific Cryptosporidium parvum antigens. Clin Microbiol Rev. 1999 , 37(5):1385-92).

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Abstract

The invention discloses a cryptosporidium parvum recombinant antigen gene (i) MUCIN (/i) and an expressed fusion protein recombinant antigen rMUCIN. 520 parts of healthy human serum are randomly taken, the recombinant antigens rMUCIN and rCp23 are used as detection antigens, a human serum IgG antibody is detected by ELISA (enzyme-linked immunosorbent assay), spss software is used for analysis of data, and the results show that the rMUCIN protein and the rCp23 protein have the same positive rate in detection of unknown serum (XC2=0.370, and P=0.543>0.05), while the positive rate of the rMUCIN protein in detection of the unknown serum is higher than that of a cryptosporidium parvum crude antigen (XC2=5.222, and P=0.02<0.05). The invention further provides a kit for ELISA detection of the anti-cryptosporidium parvum IgG antibody, which takes the recombinant antigen rMUCIN as the detection antigen, takes the rCp23 as a positive control and has stronger specificity, sensitivity and reliability.

Description

technical field [0001] The invention belongs to the technical field of biological diagnosis, and specifically relates to a cryptosporidium parvum recombinant antigen for diagnosis and a kit for detecting anti-Cryptosporidium parvum IgG antibody using the antigen as a diagnostic antigen. Background technique [0002] Cryptosporidium is apicomplexan protozoa. From the aspect of pathogenicity, the worm belongs to opportunistic protozoa. The worms mainly parasitize the microvilli of the epithelial cells of the gastrointestinal tract, and the symptoms after infecting the host vary in severity. Cryptosporidium is recognized as one of the important causes of diarrhea in children in developing countries and is also the main cause of death for AIDS patients and those with low immunity. So far, Cryptosporidium is divided into 24 species, of which the main species infecting humans is Cryptosporidium parvum ( C. parvum ) and Cryptosporidium hominis ( C. hominis ). The United Sta...

Claims

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Application Information

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IPC IPC(8): C12N15/30C07K14/44G01N33/569
Inventor 尹继刚高依然向梅姜宁陆慧君陈启军
Owner JILIN UNIV
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