旋毛虫钙网蛋白的表达和生物学特性鉴定

Expression and biological characteristics of a novel calreticulin from Trichinella spiralis

目的 克隆表达旋毛虫钙网蛋白(TsCRT),并对其生物学特性进行鉴定。方法 通过RT-PCR扩增TsCRT基因,构建重组质粒pMD19T-TsCRT,测序正确后亚克隆至表达载体pQE80L,构建重组表达载体pQE80L-TsCRT,转化大肠埃希菌BL21(DE3),以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,通过Western blot、qPCR和免疫荧光试验(IFT)鉴定rTsCRT的反应原性及其在不同虫期的表达与组织定位,通过动物免疫试验观察rTsCRT的免疫原性。结果 成功构建重组表达载体pQE80L-TsCRT,转化DE3后经IPTG诱导表达相对分子质量为47×10^(3)的rTsCRT蛋白。用纯化的rTsCRT免疫小鼠3次,血清抗体效价达1∶10^(5)。Western blot显示,rTsCRT可被抗rTsCRT血清和旋毛虫感染小鼠血清识别。qPCR检测显示,TsCRT在旋毛虫肌幼虫、肠道感染性幼虫、成虫和新生幼虫期均有转录和表达。IFT试验显示,TsCRT主要定位在旋毛虫的表皮和胚胎中。结论 TsCRT在旋毛虫不同虫期均有表达,重组表达的rTsCRT具有良好的抗原性,可作为制备抗旋毛虫疫苗及血清学诊断的候选靶抗原。

Objective To express a Trichinella spiralis calreticulin(TsCRT) gene and identify its biological characteristics. Methods The TsCRT gene was amplified by RT-PCR and the recombinant plasmid(pMD19T-TsCRT) was constructed. The TsCRT gene in recombinant plasmid was sub-cloned into the prokaryotic expression vector pQE80L and recombinant expression plasmid pQE80L-TsCRT was prepared. The pQE80L-TsCRT was transformed into an E. coli BL21(DE3),and induced by IPTG. The reactogenicity of rTsCRT,expression and tissue localization of TsCRT at various T. spiralis stages was investigated by Western blot, qPCR and immunofluorescence test(IFT). The immunogenicity of rTsCRT was assessed by immunization of mice with rTsCRT. Results Recombinant TsCRT(rTsCRT) with relative molecular weight of 47×10^(3) was expressed by pQE80L-TsCRT constructed in this study. Serum anti-rTsCRT antibody titer of immunized groups was up to 1∶10^(5) after the third immunization. Western blot results showed that rTsCRT could be recognized by anti-rTsCRT immune serum and T. spiralis-infected mouse serum. TsCRT gene was transcribed and expressed at all various T. spiralis stages(muscle larvae, intestinal infective larvae, adult worms and newborn larvae). The IFT results revealed that TsCRT was principally localized in out cuticle and intrauterine embryos of female adults. Conclusion TsCRT has good antigenicity and can be regarded as the candidate target antigen for anti-Trichinella vaccines and serodiagnosis.