论文部分内容阅读
目的体外扩增日本血吸虫中国大陆株脂肪酸结合蛋白(SjFABPc)抗原的编码区基因序列,将其克隆到真核表达质位pcDNA3中,为进一步对其进行核酸疫苗研究奠定基础。方法特定寡核苷酸引物的设计与合成;异硫氰酸胍-酚-氯仿一步法分离日本血吸虫成虫RNA,RT-PCR扩增目的基因;分子克隆常规操作将扩增产物亚克隆至中间载体pUC18中,然后走向克隆到真核表达载体pcDNA3中。结果RT-PCR特异性扩增出SjFABPc编码区基因序列,其片段大小为440hp;经酶切、PCR鉴定表明所构建的质粒pUC-SjFABPc和pCD-SjFABPc中含有所扩增的基因序列。结论RT-PCR扩增的SjFABPc抗原编码区基因序列与预期长度相村合;成功地构建了含目的基因的真核表达质粒pCD-SjFABPc。从而,为进一步对其进行DNA免疫系列研究工作奠定了基础。
Objective To amplify the coding region of the SjFABPc antigen of Schistosoma japonicum in vitro and cloned it into the eukaryotic expression plasmid pcDNA3 to lay the foundation for its further study on nucleic acid vaccine. Methods Design and synthesis of specific oligonucleotide primers; Isolation of adult Schistosoma japonicum RNA by one-step guanidinium thiocyanate-phenol-chloroform method, amplification of the target gene by RT-PCR; Molecular cloning routinely subcloned the amplified product into an intermediate vector pUC18, and then cloned into the eukaryotic expression vector pcDNA3. Results The gene sequence of SjFABPc coding region was amplified by RT-PCR. The fragment size was 440hp. The recombinant plasmid pUC-SjFABPc and pCD-SjFABPc were identified by restriction enzyme digestion and PCR. Conclusion The coding sequence of SjFABPc antigen amplified by RT-PCR was in accordance with the expected length. The eukaryotic expression plasmid pCD-SjFABPc containing the target gene was successfully constructed. Therefore, it laid the foundation for further research on DNA immunology.