【摘要】 目的制备重组刚地弓形虫微线体蛋白10(MIC10),并对其免疫学特性进行分析。方法利用逆转录PCR(RT-PCR)技术从弓形虫总RNA中反转录制备第一链cDNA,再利用特异性引物从cDNA中扩增出编码MIC10的基因片段,构建重组表达质粒pET28a-MIC10,转化入大肠埃希菌BL21(DE3)后用异丙基硫代半乳糖苷(IPTG)进行诱导表达,通过镍螯合亲和层析法纯化重组MIC10蛋白。用纯化的重组蛋白免疫家兔,制备抗MIC10多克隆抗体,采用Western blot分析重组MIC10蛋白的免疫学特性。结果采用RT-PCR法从弓形虫cDNA中反转录扩增到长度约为416bp的特异性DNA,基因序列分析证实为编码MIC10的基因片段;制备的重组表达质粒pET28a-MIC10转化大肠埃希菌BL21(DE3)后经IPTG诱导,能表达重组MIC10蛋白;用纯化的重组MIC10蛋白免疫家兔,血清抗体效价达到1∶200 000以上。Western blot分析显示该抗体能识别弓形虫排泄分泌抗原中的天然MIC10分子而不识别弓形虫成虫抗原,证明MIC10为外分泌蛋白。结论制备了具有天然免疫原性的重组弓形虫MIC10蛋白,并证明该蛋白为外分泌蛋白。更多还原

【Abstract】 Objectives To prepare a recombinant microneme protein 10(MIC10)fromToxoplasma gondii and to analyze its immunological characteristics. Methods Reverse transcription PCR(RT-PCR)was first used to prepare firststrand cDNA from Toxoplasma total RNA,and then the first-strand cDNA was used as a template to amplify the DNA fragment encoding the MIC10 mature peptide with gene-specific primers.This gene fragment was cloned into the plasmid pET28 a(+)to construct the recombinant expression plasmid pET28 a-MIC10.The recombinant expression plasmid pET28 a-MIC10 was transfected into E.coli BL21(DE3)competent cells to develop an engineered bacterium.Isopropylβ-D-1-thiogalactopyranoside(IPTG)was used to induce positive clones of the engineered bacterium to express the recombinant MIC10 protein.The purified recombinant MIC10 protein was prepared using nickel chelating affinity chromatography.A rabbit was immunized with the purified recombinant MIC10 protein to prepare IgG polyclonal antibodies against the MIC10 protein.The immunological characteristics of the recombinant MIC10 protein were analyzed using Western blotting. Results A specific DNA band of 416 bp was amplified from the first-strand cDNA of T.gondii,and the results of DNA sequencing verified that this DNA fragment is the gene encoding the MIC10 protein of T.gondii.IPTG induced the engineered bacterium containing the recombinant expression plasmid pET28 a-MIC10 to express the recombinant MIC10 protein.The titer of serum IgG antibodies against the recombinant protein MIC10 in a rabbit immunized with the pure recombinant MIC10 protein was higher than 1:200 000.The natural MIC10 protein of the excreted and secretory antigen(ESA)of T.gondii was recognized by the purified antibody IgG against the recombinant MIC10 protein,which further confirmed that MIC10 is an exocrine protein. Conclusion Recombinant MIC10 protein from T.gondii was successfully prepared with natural immunogenicity,and findings have indicated that this protein is an exocrine protein.更多还原