Cloning And Expression Of Triosephosphate Isomerase Protein From Dermatophagoides Farina And Allergenicity Identification

Objective:To express and purify the recombinant triosephosphate isomerase protein in the prokaryotic expression system and identify the recombinant protein for allergenicity characteristics.Methods:① Triosephosphate Isomerase was amplified by RT-PCR after the total RNA of dermatophagoides farinae was extracted. ②The recombinant plasmid were transformed into E.coli Top10 following PCR products and p UC57 being connected.Then the recombinant E.coli Top10 strain was cultrured in LB media with ampicillin(100 mg/L) at 37℃. Plasmid was extracted and identificated after being cut by double enzyme Bam H I and Eco R I. The gene fragment of triosephosphate Isomerase protein was synthesized and sub-cloned in p ET-32 a vector. ③Using biology software predict bioinformatics analysis of the triosephosphate isomerase protein. Base on the analysis to construct evolutionary tree by the software. ④ Recombinant triosephosphate Isomerase protein was induced to express IPTG in E.coli BL21.Products was expressed and purified successively by denaturation, renaturation, Ni-NTA chromatography and Superdex 75 gel filtration chromatography in order to get allergenicity of the recombinant protein. Western blotting tests immunogenicity of recombinant triosephosphate isomerase protein. ⑤Western blotting、ELISA results showed that 4 casesof serum ofdermatophagoides farina allergic- patients from the affiliated hospital of guangzhou medical college clinical skin test took on strong positive reaction, 2 cases of healthy control group prove negative.The skin prink test to detect the positive rate of 44 cases of powder dust mite extract triosephosphate Isomerase protein.Results:①The result to construct recombinant cloning vector and expression vector was proved to be successful.Specific bands appear in the expected position indicate the gene segment consistent with the expected size. ② Biological software analyses triose phosphate isomerase protein is hydrophobic protein which amino acid number is 247, molecular weight is 27134. ③ To get high purity of recombinant triosephosphate isomerase after renaturation and purification, products were highly expressed as inclusion bodies by prokaryotic expression system and recombinant proteins can be combined with resistance His- tag monoclonal antibody specificity.④ The triosephosphate isomerase protein showed Ig E-binding on immunoblot with the sera of all 4(100%) dermatophagoides farina-allergic patients tested,but was not bound by Ig E antibodies in sera obtained from any of2non-allergic healthy control.⑤ In vivo testing of recombinant triosephosphate isomerase protein with dermatophagoides farina-allergic patients showed skin reactivity in14 of 44 patients.Conclusions: The triosephosphate isomerase protein was discovered and identified as a major dust mite allergen, the structure of the triosephosphate isomerase protein was analyzed by biology software and recombinant protein has strong allergenicity.