论文部分内容阅读
目的构建日本血吸虫天然分子疫苗相关靶基因的双价疫苗pVAX1/SjRPS4·CB、pcDNA3.0/SjRPS4·CB,并观察其免疫保护效果。方法分别以pcDNA3.0/SjRPS4、pcDNA3.0/SjCB为模板,设计引物扩增SjRPS4、SjCB,重叠PCR法将SjRPS4、SjCB拼接,扩增,经双酶切后与真核质粒载体pVAX1、pcDNA3.0连接,转化DH5α细胞,筛选阳性克隆;提取双价重组质粒、空质粒及pVAX1/SjRPS4,经左腿股四头肌注射昆明小鼠,14d后取肌肉组织切片进行间接免疫荧光及酶免疫组织化学染色,证实双价重组质粒的表达;提取pVAX1/SjRPS4·CB、pcDNA3.0/SjRPS4·CB、pcDNA3.0/SjRPS4、pcDNA3.0/SjCB及相应空载体,100μg/只或等量PBS经左腿股四头肌注射昆明小鼠,4周后以(20±1)条尾蚴贴腹感染,6周后检测减虫率、减卵率。结果PCR、EcoRI/XhoI双酶切及测序证实双价疫苗pVAX1/SjRPS4·CB、pcDNA3.0/SjRPS4·CB构建成功;肌肉组织间接免疫荧光及酶免疫组织化学染色证实重组质粒能够在小鼠肌肉细胞内成功表达;与PBS组相比,pVAX1/SjRPS4·CB、pcDNA3.0/SjRPS4·CB、pcDNA3.0/SjRPS4、pcDNA3.0/SjCB免疫小鼠各组间差异均有统计学意义(P<0.05),各组与相应的空质粒组相比各指标差异亦具有统计学意义(P<0.05);两种不同载体构建的双价疫苗之间比较差异则无统计学意义(P>0.05)。结论成功构建真核双价重组质粒pVAX1/SjRPS4·CB、pcDNA3.0/SjRPS4·CB,二者免疫昆明小鼠均可以产生比单价疫苗更好的免疫保护效果。
Objective To construct the bivalent vaccine pVAX1 / SjRPS4 · CB, pcDNA3.0 / SjRPS4 · CB of Schistosoma japonicum natural molecular vaccine-associated target genes and observe its immunoprotective effect. Methods SjRPS4 and SjCB were amplified by PCR using pcDNA3.0 / SjRPS4 and pcDNA3.0 / SjCB as template. SjRPS4 and SjCB were spliced and amplified by overlapping PCR. After double digestion, they were ligated with eukaryotic plasmid vector pVAX1, pcDNA3 .0 were ligated and transformed into DH5α cells, and the positive clones were screened. The double-stranded recombinant plasmids, empty plasmids and pVAX1 / SjRPS4 were extracted and transfected into Kunming mice via quadriceps muscles of left leg. After 14 days, the muscle sections were harvested for indirect immunofluorescence and enzyme immunoassay Histochemical staining confirmed the expression of bivalent recombinant plasmid; pVAX1 / SjRPS4 · CB, pcDNA3.0 / SjRPS4 · CB, pcDNA3.0 / SjRPS4, pcDNA3.0 / SjCB and corresponding empty vector, 100μg / Kunming mice were injected with the left quadriceps quadriceps muscles. After 4 weeks, the mice were infected with (20 ± 1) cercariae and the worm reduction rate and the oviposition rate were measured after 6 weeks. Results The divaporial vaccine pVAX1 / SjRPS4 · CB and pcDNA3.0 / SjRPS4 · CB were successfully constructed by double enzyme digestion and sequencing with PCR and EcoRI / XhoI. The results of indirect immunofluorescence and enzyme immunohistochemistry confirmed that the recombinant plasmids could be expressed in mouse muscle SjRPS4 · CB, pcDNA3.0 / SjRPS4 · CB, pcDNA3.0 / SjRPS4, pcDNA3.0 / SjCB immunized mice were significantly higher than those in PBS group (P (P <0.05). There was also a statistically significant difference between each group and the corresponding empty plasmid group (P <0.05). There was no significant difference between the two bivalent vaccines (P> 0.05 ). Conclusions The eukaryotic divalent recombinant plasmids pVAX1 / SjRPS4 · CB and pcDNA3.0 / SjRPS4 · CB were successfully constructed, and both immune Kunming mice could produce better immunoprotective effect than the monovalent vaccine.