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为开发新型重组减毒鼠伤寒沙门菌口服活疫苗载体,本研究以pYA3493质粒为基础,用鼠伤寒沙门菌sopE_(Nt100)基因及其启动子替代原有的P_(trc)启动子,构建沙门菌三型分泌表达载体pYA-sopE_(Nt100);再将质粒pYA-sopE_(Nt100)电转入沙门菌ΔcrpΔasd SL1344,构建减毒鼠伤寒沙门菌ΔcrpΔasd SL1344(p YA-sop E_(Nt100))三型分泌表达系统,研究其生物学特性,进一步将报告基因egfp克隆入sop E基因下游,构建重组菌株ΔcrpΔasd SL1344(p YA-sop E_(Nt100)-egfp),感染Vero细胞,用Western blotting分析该系统递呈外源抗原的能力。PCR、酶切及测序结果表明,减毒鼠伤寒沙门菌ΔcrpΔasd SL1344(p YA-sop E_(Nt100))三型分泌表达系统构建成功;生物学特性鉴定结果表明,其血清型与亲本株Δcrp SL1344及野生株SL1344保持一致;其生化特性与亲本株基本相近,但与野毒株相比发生明显变化;生长速度也更为缓慢;重组菌株ΔcrpΔasd SL1344(p YA-sop E_(Nt100))的LD50较野生株SL1344降低了7.0×104倍;Western blotting结果发现,重组菌培养上清中能检测到Sop E_(Nt100)-egfp融合蛋白(37 k Da);重组菌株感染Vero细胞后,可以同时检测到Sop E_(Nt100)-egfp融合蛋白(37 k Da)和EGFP蛋白(27 k Da)。以上结果证实,本研究成功构建了新型减毒鼠伤寒沙门菌ΔcrpΔasd(p YA-sop E_(Nt100))三型分泌表达系统,其能够有效递呈外源抗原,该重组菌株有潜力作为安全、稳定、高效表达外源基因的口服重组活疫苗载体。
In order to develop a new recombinant attenuated Salmonella typhimurium oral live vaccine vector, based on the pYA3493 plasmid, Salmonella typhimurium sopE_ (Nt100) gene and its promoter were used to replace the original P trc promoter to construct Salmonella The plasmid pYA-sopE_ (Nt100) was electroporated into Salmonella ΔcrpΔasd SL1344 to construct an attenuated Salmonella typhimurium ΔcrpΔasd SL1344 (p YA-sop E_ (Nt100)) tris Type secretory expression system and study its biological characteristics. The reporter gene egfp was further cloned into the downstream of sop E gene and the recombinant strain ΔcrpΔasd SL1344 (pYA-sop E_ (Nt100) -egfp) was infected and infected with Vero cells. The ability of the system to deliver exogenous antigens. The results of PCR, restriction enzyme digestion and sequencing showed that the triplex secretion expression system of attenuated Salmonella typhimurium ΔcrpΔasd SL1344 (pYA-sop E_ (Nt100)) was constructed successfully. The biological characteristics showed that the serotypes of the three strains were compared with the parent strain Δcrp SL1344 And the wild type strain SL1344. The biochemical characteristics of the recombinant strain ΔcrpΔasd SL1344 (pYA-sop E_ (Nt100)) were similar to those of the parental strain but significantly different from those of the wild-type strain. The LD50 of the recombinant strain ΔcrpΔasd SL1344 The wild-type strain SL1344 was reduced by 7.0 × 104 times. Western blotting showed that Sop E_ (Nt100) -egfp fusion protein (37 kDa) was detected in the supernatant of the recombinant strain. After the recombinant strain was infected with Vero cells, Sop E_ (Nt100) -egfp fusion protein (37 kDa) and EGFP protein (27 kDa). The above results confirmed that the present invention successfully constructed a novel type III secretion expression system of attenuated Salmonella typhimurium ΔcrpΔasd (pYA-sop E_ (Nt100)), which can effectively present exogenous antigens and has the potential as a safe, Orally recombinant live vaccine vectors that stably and efficiently express foreign genes.