Toxophasma gondii detecting kit based on recombined antigen

A technology of Toxoplasma gondii and a kit, which is applied in the field of Toxoplasma gondii detection kits, can solve the problems of high toxicity of host bacteria, lack of modification function, no specific immunoreactivity, etc., and achieves the effect of good immune activity

Inactive Publication Date: 2006-11-15
深圳市绿诗源生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Prokaryotic expression systems (such as Escherichia coli) lack the post-translational modification function of proteins, so most of the recombinant SAG1 proteins obtained in Escherichia coli in the past form inclusion bodies, and the expression products do not have specific immunoreactivity due to misfolding. Denaturation and renaturation process can restore partial activity
In addition, when the whole gene of SAG1 is cloned into Escherichia coli, the expression level is not high due to the high toxicity to the host bacteria.

Method used

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  • Toxophasma gondii detecting kit based on recombined antigen
  • Toxophasma gondii detecting kit based on recombined antigen
  • Toxophasma gondii detecting kit based on recombined antigen

Examples

Experimental program
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Effect test

Embodiment 1

[0074] Example 1 Construction of pET32a(+)-tSAG1

[0075] Extract the recombinant plasmid DNA of the transformed bacteria, perform 1% agarose electrophoresis with the empty plasmid DNA, and then carry out single- and double-enzyme digestion for identification. The pET32a(+)-tSAG1 positive recombinant plasmid can be digested with Nco I and the size is about 6600bp The left and right single bands can produce two bands of about 5900bp and 700bp after double digestion with Nco I+HindIII, which is consistent with the expected results. However, pET32a (+) has only a band of 5900bp after Nco I single enzyme digestion, the above results show that the pET32a (+)-tSAG1 recombinant plasmid ( figure 1 ).

Embodiment 2

[0076] Example 2 Induced expression of engineering bacteria pET32a(+)-tSAG1 / BL21

[0077] After pET32a(+)-tSAG1 / BL21 was induced by IPTG, a specific expression band appeared at about 40kDa by SDS-PAGE analysis, which basically coincided with the expected molecular weight. The empty vector expressed thioredoxin (Thioredoxin, Trx) at the position of 21kDa, while the empty vector and the recombinant plasmid had no specific band of expressed protein before induction. After the recombinant bacteria were broken by ultrasonic waves, they were centrifuged at 14,000rpm for 20min, and the supernatant and precipitate were collected for SDS-PAGE. The results showed that the target protein was mainly expressed in soluble form. The target protein accounted for 34.36% of bacterial protein. ( figure 2 )

Embodiment 3

[0078] Purity of rSAG1 after Ni column purification of embodiment three

[0079] A large amount of recombinant protein is expressed under optimized conditions. After ultrasonic destruction, most of the recombinant protein exists in the ultrasonic supernatant in a soluble form. After the recombinant protein in the supernatant is purified by Ni-NTA, the purity can reach more than 60% %, the purified recombinant protein can obtain a purity of more than 90% after being purified by Sephadex-G75 (Fig. 3).

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Abstract

A reagent kit based on recombinant antigen for detecting Toxoplasma is prepared through taking 542-1218 fragment (t SAG1) from the primary surface antigen gene SAG1 of Toxoplasma, subcloning it to soluble expression carrier pET32a(+), transferring it to colibacillus, configuring engineering bacterium pET32a-tSAG1/BL21, IPTG induced efficient expression, ultrasonic splitting to obtain supernatant, purifying by Ni-NTA and Sephadex-G75, and coating the microholes on ELISA plate. Its test paper can also be prepared by same way.

Description

technical field [0001] The present invention relates to a detection kit for Toxoplasma gondii (Toxoplasma gondii) based on recombinant antigen, which is to detect and / or diagnose Toxoplasma gondii antibody or corresponding antigen in a sample by using recombinant antigen and its corresponding monoclonal antibody, Screening and identification of this antigen or monoclonal antibody, components of the kit, detection steps and application. Background technique [0002] Toxoplasma gondii (Toxoplasma gondii) is an obligate intracellular parasitic medical protozoan, which can infect almost all warm-blooded animals (including humans), causing toxoplasmosis, and nearly 1 / 3 of the world's population is threatened. Toxoplasma gondii is an opportunistic pathogenic parasite, which usually forms cysts in people with normal immune function, causing hidden infection; but when the immune function is weakened (such as tumor patients, organ transplant patients and AIDS patients, etc.), Toxopla...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/45C12N15/63G01N33/53G01N33/577G01N33/68
Inventor 陈晓光李华
Owner 深圳市绿诗源生物技术有限公司
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