【摘要】 用日本血吸虫（Sj）成虫提取的总RNA逆转录合成CDNA第一链，并设计合成引物，经PCR扩增，扩增出26kDa的编码区基因，并进行了测序，结果表明Sj中国大陆株（SjC）、Sj菲律宾株（SjP）26kDaGST基因核苷酸同源性99．8％．然后，构建了大肠杆菌-分枝杆菌穿梭表达质粒pBCG－Sj26Ⅰ-Ⅳ．再将其依次分别转化大肠杆菌、耻垢分枝杆菌和BCG中，筛选出重组耻垢分枝杆菌疫苗和重组BCG多价疫苗．经SDS－PAGE和薄层扫描分析表明，Sj26kDaGST的表达效率在大肠杆菌、耻垢分校杆菌和BCG中分别占菌体总蛋白的25％-37％、10％－28％、10％－15％．通过酶切图谱、PCR技术和Westernblot分析，证实构建的Sj26kDa基因工程BCG多价疫苗是成功的．用其免疫BABL／c小鼠，结果表明它对日本血吸虫的感染有明显的预防作用，重组BCG多价疫苗的减虫率达27．36％（t=3．42，P＜0．01），减卵率达60．50％（t=6．19，P＜0．01）．实验组虫卵肉芽肿平均面积以及免疫后血清抗体水平与对照组相比其差别具有显著意义．该研究为日本血吸虫成虫疫苗的研究提供了一条新的途径．更多还原

【Abstract】 Three E. coli-Mycobacteric shuttle plasmids including pBCG-2000, pME-65 and pBCG-2100 were constructed. Sj26kDa GST cDNA was obtained by RT-PCR from Sj26 mRNA ofSchistosona japonicum adults and pGEX as templets while heat shock protein (hsp )70promotor was done with pMT-70 as templet. The results of sequences analyses indicatedthat the nucleotide sequences show 99. 8% identity comparing the Chinese and Philipping S-japonicum- After Sj26 GST genes was cloned into the downstream of hsp70 promotor ofpMT-70 the vectors of pMT-Sj26 was constructed, The fused genes of hsp70 promotor-Sj26was cut from the vecter, Then E. coli-MycObacteria shuttle expression p1asmids pBCG-Sj26Ⅰ -Ⅳ was constructed by cloning the fused genes into the pBCG-2000. The recombinantM. smegmatis vaccine and recombniant BCG multivalent vaccine were screened after transfor-mation of pBCG-Sj26Ⅰ -Ⅳ into E. coli and M. smegmatis mc2155 and BCG. The results ofSDS-PAGE and thin-layer scanning analyses showed that the expression efficiency ofSj26kDa GST was about 25%-37%, 10%-28% and 10%-15% of total bacteria protein in E.coli, M, smegmatis and BCG respectively. The construction of genetic engineering BCG mul-tivalent vaccine of Sj26kDa was identified by restrictive nuclease spectrum, PCR and West-ern blot. Prevention from attacking of S. japonicum was significant by immunizing BALB/cmice with the vaccine. The ratio of decreasing adult worms and the ratio of decreasing eggswere 27. 36% (P<0. 01 ) and 60. 50% (P<0. 01 ) respectively by recombinant BCG vaccine.The average area of egg granulomas and level of antibody have significant difference betweenexperimental groups and control. The study found a new way for the research of anti-S.japonicum vaccine.更多还原