日本血吸虫组织蛋白酶B表达载体的构建及其核酸疫苗对小鼠保护性免疫的研究

发布时间：2018-08-07 09:45

【摘要】：目的:构建日本血吸虫组织蛋白酶B(Sjcb2)原核表达载体pWR450-1/sjcb2并表达;构建真核表达载体pcDNA3.1(+)/sjcb2,转染HeLa细胞,并将pcDNA3.1(+)/sjcb2直接肌注BALB/c小鼠,观察其所诱导的体液免疫和细胞免疫应答水平,初步研究该核酸疫苗对BALB/c小鼠诱导的免疫保护性作用,为研制新型抗日本血吸虫感染核酸疫苗提供实验依据。方法:用PRIMER5.0引物设计软件自行设计引物,PCR法扩增sjcb2基因片段,将PCR产物纯化后与pUCm-T载体连接,经蓝白斑筛选、双酶切分析和PCR鉴定后,亚克隆入pWR450-1原核表达载体及pcDNA3.1(+)真核表达载体中,经抗生素(氨苄青霉素)筛选、双酶切鉴定、PCR鉴定及测序鉴定后,将构建的pWR450-1/Sjcb2重组原核表达载体转化大肠杆菌后经IPTG诱导表达,SDS-PAGE及Western blotting鉴定表达情况。构建的pcDNA3.1(+)/sjcb2重组质粒电穿孔转染HeLa细胞,免疫细胞化学法观察其在真核细胞中的表达情况;将核酸疫苗pcDNA3.1(+)/Sjcb2注射到BALB/c小鼠后腿股四头肌肌肉组织中,每2周免疫一次,共免疫3次。末次免疫后2周,用日本血吸虫尾蚴攻击感染BALR/c小鼠。PCR及免疫组化法分析Sjcb2基因在小鼠体内的稳定性及表达情况;MTT法检测小鼠脾淋巴细胞特异性增殖反应;ELISA双抗体夹心法测定攻击感染前后小鼠脾淋巴细胞培养上清中IFN-γ和IL-4的水平;琼脂双向扩散试验测定小鼠血清中Sjcb2抗体水平;攻击感染42天后剖杀小鼠,计算小鼠所荷成虫对数及肝脏所荷虫卵数。结果:成功构建pWR450-1/Sjcb2重组原核表达载体,并在E.coli DH5α中表达出一分子量约为86KDa的重组融合蛋白。成功构建pcDNA3.1(+)/sjcb2真核表达重组体,电穿孔转染HeLa细胞,免疫细胞化学法表明Sjcb2能在HeLa细胞胞浆中表达。将核酸疫苗pcDNA3.1(+)/Sjcb2
[Abstract]:Objective: to construct the prokaryotic expression vector pWR450-1/sjcb2 of schistosoma japonicum cathepsin B (Sjcb2), construct eukaryotic expression vector pcDNA3.1 () / sjcb2, transfect it into HeLa cells, and inject pcDNA3.1 () / sjcb2 directly into BALB/c mice, and observe the level of humoral and cellular immune response induced by pcDNA3.1 () / sjcb2. The immune protective effect of the nucleic acid vaccine on BALB/c mice was preliminarily studied, which provided experimental basis for the development of a novel nucleic acid vaccine against Schistosoma japonicum infection. Methods: the sjcb2 gene fragment was amplified by using PRIMER5.0 primer design software. The PCR product was purified and ligated with pUCm-T vector. The product was screened by blue and white spot and identified by double enzyme digestion and PCR. Subcloned into pWR450-1 prokaryotic expression vector and pcDNA3.1 () eukaryotic expression vector, screened by antibiotics (ampicillin), identified by double enzyme digestion and identified by PCR and sequencing. The recombinant prokaryotic expression vector of pWR450-1/Sjcb2 was transformed into E. coli and identified by SDS-PAGE and Western blotting induced by IPTG. The pcDNA3.1 () / sjcb2 recombinant plasmid was electroporated into HeLa cells and its expression in eukaryotic cells was observed by immunocytochemistry. The nucleic acid vaccine pcDNA3.1 () / Sjcb2 was injected into the quadriceps muscle of the hind leg of BALB/c mice and immunized every 2 weeks. Total immunization 3 times. 2 weeks after the last immunization, The stability and expression of Sjcb2 gene in mice infected with Schistosoma japonicum cercariae in vivo were analyzed by .PCR and immunohistochemical method. The specific proliferative reaction of spleen lymphocytes in mice was detected by MTT assay and the attack was detected by Elisa double antibody sandwich method. The levels of IFN- 纬 and IL-4 in the culture supernatant of spleen lymphocytes of mice before and after infection; Agar bidirectional diffusion test was used to determine the level of Sjcb2 antibody in serum of mice, and after 42 days of infection, mice were killed to calculate the logarithm of adult worms and the number of eggs loaded by liver. Results: the recombinant prokaryotic expression vector of pWR450-1/Sjcb2 was successfully constructed, and a recombinant fusion protein with molecular weight of 86KDa was expressed in E.coli DH5 伪. The eukaryotic expression recombinant of pcDNA3.1 () / sjcb2 was successfully constructed and transfected into HeLa cells by electroporation. Immunocytochemistry showed that Sjcb2 could be expressed in the cytoplasm of HeLa cells. Nucleic acid vaccine pcDNA3.1 () / Sjcb2
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R383

【共引文献】