日本血吸虫SjOST48基因的克隆、表达及其重组蛋白免疫保护效果分析

Cloning, expression of gene SjOST48 from Schistosoma japonicum and evaluation of the immunoprotective efficacy of rSjOST48 in mice

摘要:

为了鉴定日本血吸虫SJCHGC01743基因并评估其重组蛋白作为新的血吸虫病候选疫苗抗原的潜力，利用PCR技术扩增日本血吸虫SJCHGC01743基因，应用荧光实时定量PCR分析该基因在日本血吸虫不同发育阶段虫体的转录水平，以pET-28a(+) 为载体构建重组表达质粒并诱导其在大肠杆菌中表达。将纯化的重组蛋白免疫BALB/c小鼠制备免疫血清，利用Western blotting检测重组蛋白的免疫原性，应用间接免疫荧光技术对SJCHGC01743进行蛋白组织定位，利用间接ELISA方法检测小鼠血清中特异性抗体水平。将重组抗原免疫小鼠，评估其免疫保护效果。PCR扩增得到1 248 bp不含信号肽的cDNA序列，同源性分析结果显示，该基因为日本血吸虫寡糖转移酶OST48亚基，命名为SjOST48。实时定量PCR分析显示SjOST48在检测的童虫和成虫各个期别虫体中均有转录，其中在28 d虫体中的转录水平最高，在42 d雌虫中的转录量显著高于雄虫。构建的重组表达质粒pET-28a(+)-SjOST48成功在大肠杆菌中表达，重组蛋白rSjOST48分子量约50 kDa。Western blotting分析表明rSjOST48能被小鼠免疫血清识别，具有良好的免疫原性，间接免疫荧光实验表明SjOST48蛋白主要分布于虫体体被，少量分布于实质。ELISA检测结果表明rSjOST48能诱导产生较高的特异性IgG、IgG1和IgG2a抗体。动物免疫保护实验结果表明SjOST48能诱导小鼠产生32.62% (P<0.05) 的减虫率及57.61% (P<0.01) 的肝脏减卵率。本研究为深入探讨日本血吸虫SjOST48基因的生物学功能及筛选新的血吸虫疫苗候选分子奠定了基础。

Abstract:

To identify SJCHGC01743 gene of Schistosoma japonicum and evaluate the potential of the recombinant protein as a new vaccine candidate for schistosomiasis, polymerase chain reaction (PCR) technique was used to amplify the cDNA of the gene and real-time RT-PCR was used to analyze the transcription profiles of SJCHGC01743 at different development stages. Recombinant plasmid was successfully constructed and transformed into competent Escherichia coli BL21 (DE3). Then the recombinant protein was expressed, purified and emulsified with ISA206 adjuvant to immunize BALB/c mice for three times. The immunogenicity was confirmed by Western blotting and tissue localization was detected by indirect immunofluorescent assay. The specific antibody level was detected by ELISA. The immunoprotection of rSjOST48 was evaluated by the reduction in worm and egg counts in mice. A cDNA with 1 248 nucleotides was isolated from 28-day-old schistosomes cDNAs by PCR. Sequence analysis revealed that SJCHGC01743 was a 48-kDa subunit of the oligosaccharyltransferase complex (OST48) and named as SjOST48. Real-time PCR analysis indicated that this gene was expressed in all investigated stages and had the highest expression level in 28 d worms, the level of gene transcription in female worms was significantly higher than that of male worms. Then recombinant plasmid pET28a(+)-SjOST48 was successfully constructed and expressed in E. coli BL21 (DE3). Western blotting analysis showed that rSjOST48 had good immunogenicity. Indirect immunofluorescent analysis revealed that SjOST48 was mainly distributed on the tegument of the worms. The result of ELISA indicated that the rSjOST48 vaccinated group could induce a significant increase in the level of specific IgG, IgG1 and IgG2a. An immunoprotection experiment showed that the vaccination of rSjOST48 in mice induced 32.62% (P<0.05) reduction in the numbers of worms and 57.61% (P<0.01) in eggs in liver, compared with that of the control group. This study provides the foundation for proceeding further research on the biological function of SjOST48 and screening new vaccine candidates for schistosomiasis.