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以贾第虫的c DNA作为模板,进行PCR扩增得到目的片段,克隆入p VAX1载体,构建重组质粒p VAX1-α1-giardin;以电击转化的方式转入减毒沙门菌株SL7207,构建重组沙门菌SL7207/p VAX1-α1-giardin;通过体外连续培养测定重组菌株的载体携带稳定性;30只清洁级雌性BALB/c小鼠,随机分为3组,每组10只,分别以1×106的SL7207/p VAX1-α-giardin、SL7207/p VAX1和无菌1×PBS(p H 7.0)口服免疫小鼠,8周后处死,检测各组小鼠肠系膜淋巴结中α1-giardin的表达及血清中和小肠灌洗液中特异性Ig G和SIg A的水平。结果表明,重组减毒沙门菌SL7207/p VAX1-α1-giardin连续培养168 h后,质粒携带率为90%;口服免疫小鼠8周后,SL7207/p VAX1-α1-Giardin免疫组,肠系膜淋巴结可见α1-Giardin的高表达,血清中特异性Ig G抗体水平和小肠灌洗液中特异性SIg A水平均明显增高,分别为PBS组的3.6(P<0.01)和5.7倍(P<0.01)。本研究尝试了以减毒沙门菌为载体携带α1-giardin DNA的抗贾第虫口服疫苗的构建及免疫试验,为进一步的疫苗保护性试验和制剂研究奠定了基础。
The Giardia c DNA was used as a template for PCR amplification to obtain the target fragment and cloned into p VAX1 vector to construct the recombinant plasmid p VAX1-α1-giardin. The recombinant plasmid p VAX1-α1-giardin was transformed into attenuated Salmonella strain SL7207 by electroporation, Strain SL7207 / p VAX1-α1-giardin was determined by in vitro continuous culture. Thirty clean female BALB / c mice were randomly divided into three groups with 10 rats in each group, 1 × 106 Mice were orally immunized with SL7207 / p VAX1-α-giardin, SL7207 / p VAX1 and sterile 1 × PBS (p H 7.0) for 8 weeks and then sacrificed to detect the expression of α1-giardin in the mesenteric lymph nodes and serum Neutralize the levels of specific Ig G and SIg A in small intestinal lavage fluid. The results showed that the plasmid-carrying rate of recombinant attenuated Salmonella SL7207 / p VAX1-α1-giardin after continuous culture for 168 h was 90%. After oral administration of the vaccine for 8 weeks, the immune responses of SL7207 / p VAX1-α1-Giardin group, mesenteric lymph node The results showed that high expression of α1-Giardin, specific Ig G antibody level in serum and specific SIg A level in small intestine lavage fluid were significantly higher than those in PBS group (P <0.01) and 5.7 times (P <0.01) . In this study, the construction of the oral anti-giardia vaccine carrying alpha1-giardin DNA with attenuated Salmonella as carrier and the immunoassay were tried, which laid the foundation for further vaccine protective test and formulation research.