旋毛虫丝氨酸蛋白酶抑制因子基因的克隆、表达及抗原性研究
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摘要
旋毛虫病是由旋毛虫引起的一种危害严重的食源性人兽共患寄生虫病,可在包括人类在内的150多种动物之间广泛传播。人主要因为生食或半生食含有旋毛虫肌幼虫包囊的动物肉类而感染,严重时可以致人死亡。旋毛虫病是一个非常重要的公共卫生问题,不仅威胁着人类健康,而且给畜牧业生产造成巨大经济损失。
本研究根据GenBank数据库中旋毛虫丝氨酸蛋白酶抑制因子基因序列设计引物,从旋毛虫肌幼虫提取总RNA,应用RT-PCR扩增获得目的基因片段,进行限制性酶切后连接到表达质粒载体pET30a,构建重组表达质粒pET30a-TsSerPIN1,转化大肠杆菌DH5α,筛选阳性克隆,经PCR、限制性酶切分析及测序鉴定后,转化大肠杆菌BL21(DE3),以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。结果表明成功构建了重组表达质粒pET30a-TsSerPIN1,插入片段为1122 bp,编码373个氨基酸,与GenBank数据库中旋毛虫丝氨酸蛋白酶抑制因子基因的相似性为99%。含有pET30a-TsSerPIN1重组表达质粒的大肠杆菌BL21(DE3)诱导后得到了高效表达,SDS-PAGE分析显示表达产物分子量约为48.5 kDa,主要以包涵体形式存在。Western-blot结果表明TsSerPIN1重组蛋白可以被旋毛虫感染猪阳性血清特异性识别,具有良好的抗原性。进一步分段克隆表达了TsSerPIN基因的不同片段,结果均成功表达,对TsSerPIN1、TsSerPIN2、TsSerPIN3、TsSerPIN4和TsSerPIN5的重组蛋白进行Western-blot分析,只有TsSerPIN1和TsSerPIN5重组蛋白能被感染旋毛虫的猪血清特异性识别。TsSerPIN1和TsSerPIN5重组蛋白间接ELISA检测结果表明两种重组重组蛋白的反应原性相似,说明TsSerPIN5具有此蛋白C末端的主要抗原表位。以TsSerPIN5重组蛋白为包被抗原对实验感染旋毛虫猪血清、猪田间血清样品和健康人血清进行间接ELISA检测,表明TsSerPIN5重组蛋白具有良好的反应原性。分别应用TsSerPIN1重组蛋白和TsSerPIN5重组蛋白作为免疫原免疫小鼠后,均可诱导小鼠产生一定的免疫保护性,肌幼虫减虫率分别为40.7%和54.5%,而且可以诱导产生特异性抗体应答反应。通过研究表明,TsSerPIN5有望作为旋毛虫病免疫诊断和疫苗研制的候选抗原。
Trichinellosis caused by Trichinella spp. is a serious foodborne parasitic zoonosis and wide spread among more than 150 species of animals including human. Human can obtain this infection through ingestion of raw meat infected by Trichinella spp. and serious infection can be fatal. Trichinellosis is an important public health issue which not only threatens human health but also causes great economic losses to livestock production.
In this study the specific primers derived from T. spiralis serine proteinase inhibitor gene in GenBank database was designed and used to amplify the TsSerPIN gene from total RNA isolated from T. spiralis muscle larvae. The RT-PCR product was purified, digested and ligated into the expression vector pET30a. After transformation to E. coli DH5αand identification by PCR, restriction digestion and sequencing, pET30a-TsSerPIN1 plasmid was transformed to E. coli BL21 (DE3) and induced by IPTG for expression. The results that pET30a-TsSerPIN1 recombinant expression plasmid was successfully constructed and contained a insert of 1122 bp encoding 373 amino acids which showed 99% identity to Trichinella spp.serine proteinase inhibitor gene in GenBank database. The TsSerPIN1 recombinant protein was highly expressed in the form of inclusion body with the molecular weight of about 48.5 kDa. In Western-blot analysis the purified TsSerPIN1 recombinant protein could be specifically recognized by T. spiralis infected swine serum. Different fragments of the TsSerPIN gene were further cloned and all of the four fragments were expressed. Western-blots indicated that only TsSerPIN1 and TsSerPIN5 recombinat proteins could be recognized by T. spiralis infected swine serum. Indirect ELISA showed that TsSerPIN1 and TsSerPIN5 recombinant proteins were similar in reactivity, indicating that TsSerPIN5 had the main epitopes in the C terminal of this protein. ELISA on experimentally infected swine serum, field swine serum and human serum revealed good reactivity of TsSerPIN5 recombinant protein. After immunization of mice with TsSerPIN1 and TsSerPIN5 recombinant proteins and challenge with T. spiralis larvae, the immunized mice could get parties protection with muscle larvae reduction of 40.7% and 54.5%, respectively. and produced specific antibody response. TsSerPIN5 could be a candidate for immunodignosis and vaccines of Trichinellosis.
本研究根据GenBank数据库中旋毛虫丝氨酸蛋白酶抑制因子基因序列设计引物,从旋毛虫肌幼虫提取总RNA,应用RT-PCR扩增获得目的基因片段,进行限制性酶切后连接到表达质粒载体pET30a,构建重组表达质粒pET30a-TsSerPIN1,转化大肠杆菌DH5α,筛选阳性克隆,经PCR、限制性酶切分析及测序鉴定后,转化大肠杆菌BL21(DE3),以异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达。结果表明成功构建了重组表达质粒pET30a-TsSerPIN1,插入片段为1122 bp,编码373个氨基酸,与GenBank数据库中旋毛虫丝氨酸蛋白酶抑制因子基因的相似性为99%。含有pET30a-TsSerPIN1重组表达质粒的大肠杆菌BL21(DE3)诱导后得到了高效表达,SDS-PAGE分析显示表达产物分子量约为48.5 kDa,主要以包涵体形式存在。Western-blot结果表明TsSerPIN1重组蛋白可以被旋毛虫感染猪阳性血清特异性识别,具有良好的抗原性。进一步分段克隆表达了TsSerPIN基因的不同片段,结果均成功表达,对TsSerPIN1、TsSerPIN2、TsSerPIN3、TsSerPIN4和TsSerPIN5的重组蛋白进行Western-blot分析,只有TsSerPIN1和TsSerPIN5重组蛋白能被感染旋毛虫的猪血清特异性识别。TsSerPIN1和TsSerPIN5重组蛋白间接ELISA检测结果表明两种重组重组蛋白的反应原性相似,说明TsSerPIN5具有此蛋白C末端的主要抗原表位。以TsSerPIN5重组蛋白为包被抗原对实验感染旋毛虫猪血清、猪田间血清样品和健康人血清进行间接ELISA检测,表明TsSerPIN5重组蛋白具有良好的反应原性。分别应用TsSerPIN1重组蛋白和TsSerPIN5重组蛋白作为免疫原免疫小鼠后,均可诱导小鼠产生一定的免疫保护性,肌幼虫减虫率分别为40.7%和54.5%,而且可以诱导产生特异性抗体应答反应。通过研究表明,TsSerPIN5有望作为旋毛虫病免疫诊断和疫苗研制的候选抗原。
Trichinellosis caused by Trichinella spp. is a serious foodborne parasitic zoonosis and wide spread among more than 150 species of animals including human. Human can obtain this infection through ingestion of raw meat infected by Trichinella spp. and serious infection can be fatal. Trichinellosis is an important public health issue which not only threatens human health but also causes great economic losses to livestock production.
In this study the specific primers derived from T. spiralis serine proteinase inhibitor gene in GenBank database was designed and used to amplify the TsSerPIN gene from total RNA isolated from T. spiralis muscle larvae. The RT-PCR product was purified, digested and ligated into the expression vector pET30a. After transformation to E. coli DH5αand identification by PCR, restriction digestion and sequencing, pET30a-TsSerPIN1 plasmid was transformed to E. coli BL21 (DE3) and induced by IPTG for expression. The results that pET30a-TsSerPIN1 recombinant expression plasmid was successfully constructed and contained a insert of 1122 bp encoding 373 amino acids which showed 99% identity to Trichinella spp.serine proteinase inhibitor gene in GenBank database. The TsSerPIN1 recombinant protein was highly expressed in the form of inclusion body with the molecular weight of about 48.5 kDa. In Western-blot analysis the purified TsSerPIN1 recombinant protein could be specifically recognized by T. spiralis infected swine serum. Different fragments of the TsSerPIN gene were further cloned and all of the four fragments were expressed. Western-blots indicated that only TsSerPIN1 and TsSerPIN5 recombinat proteins could be recognized by T. spiralis infected swine serum. Indirect ELISA showed that TsSerPIN1 and TsSerPIN5 recombinant proteins were similar in reactivity, indicating that TsSerPIN5 had the main epitopes in the C terminal of this protein. ELISA on experimentally infected swine serum, field swine serum and human serum revealed good reactivity of TsSerPIN5 recombinant protein. After immunization of mice with TsSerPIN1 and TsSerPIN5 recombinant proteins and challenge with T. spiralis larvae, the immunized mice could get parties protection with muscle larvae reduction of 40.7% and 54.5%, respectively. and produced specific antibody response. TsSerPIN5 could be a candidate for immunodignosis and vaccines of Trichinellosis.
引文
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