论文部分内容阅读
目的探讨以减毒沙门菌为载体,构建表达细粒棘球蚴Eg.ferriti蛋白口服活载体疫苗的可行性。方法从细粒棘球蚴包囊中分离原头节,超声粉碎后提取总RNA,通过RT-PCR扩增细粒棘球蚴Eg.ferritin基因,然后将Eg.ferritin基因插入到pYA3341表达载体中,构建重组质粒pYA3341-Eg.ferritin,将重组质粒依次电转入减毒鼠伤寒沙门菌X3770和X4550,获得重组菌株St-Eg.ferritin,对重组菌的稳定性、安全性以及免疫原性进行评价。结果经酶切鉴定成功构建了重组质粒pYA3341-Eg.ferritin,Western blot结果证实Eg.ferritin蛋白在重组菌中获得了表达;重组菌株在体外至少传代10次而保证重组质粒不丢失;小鼠免疫试验证实,重组菌安全无毒性;重组菌口服免疫小鼠可检测到Eg.ferritin特异性IgG抗体。结论本试验成功构建了能稳定表达细粒棘球蚴Eg.ferritin蛋白的口服减毒鼠伤寒沙门菌活载体疫苗株,并显示了良好的免疫原性和安全性,为包虫病新型口服基因工程活载体疫苗的研制奠定基础。
Objective To investigate the feasibility of using live attenuated Salmonella as a vector to construct oral live carrier vaccine expressing Echinococcus granulosus. Methods The protoscoleces were isolated from the cysts of Echinococcus granulosus, and the total RNA was extracted by sonication. The Eg. Ferritin gene of Echinococcus granulosus was amplified by RT-PCR. Then, the gene of Eg. Ferritin was inserted into pYA3341 expression vector The recombinant plasmid pYA3341-Eg.ferritin was constructed and the recombinant plasmids were electroporated into attenuated Salmonella typhimurium X3770 and X4550 to obtain the recombinant St-Eg. Ferritin. The stability, safety and immunogenicity of the recombinant strain Evaluation. Results Recombinant plasmid pYA3341-Eg.ferritin was successfully constructed by restriction endonuclease digestion. Western blot showed that Eg.ferritin protein was expressed in the recombinant bacteria. The recombinant strain was passaged at least 10 times in vitro to ensure that the recombinant plasmid was not lost. Immunization of mice The test confirmed that the recombinant bacteria safe and non-toxic; recombinant E. coli vaccine can be detected Eg.ferritin specific IgG antibodies. Conclusions This study successfully constructed an oral attenuated Salmonella typhimurium live vector vaccine strain which can stably express Eg. Ferritin protein of Echinococcus granulosus, and showed good immunogenicity and safety, and was a novel oral echinococcosis gene Engineering live vector vaccine laid the foundation for the development.