摘要
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。
Aim In order to provide the basis for further study on DNA vaccine against Schistosoma jaPonicum,amp1i-fving the gene coding for SjFABPc in vitro and cloning the fragment into the eukaryotic expression vector pcDNA3. MetkodsThe design and synthesis of specific oligonuc1eotide primers 1 Tota1 RNA was isolated from adu1t worms of Chinese S' jaPon-icum using single-step method by acid guanidinium thiocyanate-phenol-chloroform (AGPC) extraction, coding region gene ofSjPABPc was amplified by RT - PCR techniqueI the fragment from RT - PCR was firstly subcloned into cloning vectorpUC18 via SacI and BamHI restriction sites and the cloned into eukaryotic expression vector pcDNA3 via EcoRI and XbaI sitesby routine molecular cloning approaches l the resulting construct was determined by PCR and restriction analysis methods. Re-sult8 The coding region of SjFABPc gene was specifical1y amplified by RT-PCR, the size of amplified fragment was 44Obase pairs 1 the cloning plasmid construct (pUC-SjFABPc) and expression plasmid (pCD-SjFABPc) contained the amplifiedfragment. Concluslon The results demonstrated that the amp1ified fragment was identical with the predicted one, the eukary-otic expression plasmid successfully constructed and provided the basis for further study on DNA immunization.
出处
《中国人兽共患病杂志》
CSCD
北大核心
1999年第3期33-36,共4页
Chinese Journal of Zoonoses
