Prokaryotic expression, purification and identification of human procalcitonin / 临床检验杂志

Objective To construct the prokaryotic expression vector of human procalcitonin(PCT) and obtain the highly purified recombinant PCT protein.Methods PCT primers were designed based on the sequence of PCT gene from NCBI,and PCT gene was amplified by PCR.Then,the recombinant vector PCT/pET-22b(+) was constructed and transferred into E.coli BL21 to induce the expression of recombinant PCT protein.Last,the recombinant PCT protein was purified by the nickel column affinity chromatography and identified by Western blot and the colloidal gold method,respectively.Results The results of agarose gel electrophoresis showed that the product of PCR amplification was about 350 bp.The homology comparison analysis revealed that the PCT gene fragment(348 bp) was successfully inserted into pTG19-T vector,and that no any base mutation was found.When the recombinant plasmid of PCT/pET-22b(+) was digested with BamH Ⅰ and HindⅢ,two pieces of about 350 bp and 5 500 bp were obtained.SDS-PAGE showed that the recombinant PCT protein with about 14 000 of Mr was existed in soluble form,and was easily obtained by the nickel column affinity chromatography.Moreover,western blot and the colloidal gold method demonstrated that the recombinant PCT protein was successfully expressed and contained histidine label(His-tag).Conclusion The recombinant vector PCT/pET-22b(+) is successfully constructed by the recombinant DNA technology,and the recombinant PCT fusion protein is successfully obtained by the nickel column affinity chromatography.

Cloning,fusion expression and identification of thioredoxin encoding gene from Toxoplasma gondii / 中国血吸虫病防治杂志

Objective To clone and express the thioredoxin(Trx)from RH strain tachyzoites of Toxoplasma gondii,estab?lish the prokaryotic expression vector and purify the recombinant protein,then produce the polyclonal anti?Trx antibody in rab?bits. Methods Trx fragment was amplified by PCR and cloned into the pET?28a(+)vector,and the recombinant protein was in?duced with IPTG and purified by Ni?NTA affinity chromatography. The polyclonal antibody specificity was detected by Western blotting. Results The trx gene was amplified from T. gondii cDNA by PCR. The recombinant plasmid trx/pET?28a(+)was use?fully constructed,and the recombinant TRX protein was expressed and purified. The TRX polyclonal antibody was also ob?tained. The specific band of TRX was detected by Western blotting. Conclusion Western blotting can detect the specificity of polyclonal anti?Trx antibody,which will facilitate the biological functions of Trx.