沙门氏菌鞭毛蛋白基因高效表达系统的构建

Establishment of the High Level Expression System for Salmonella Gene fliC i

以鼠伤寒沙门氏菌ＬＢ５０００（ｒ－ｍ＋）为第一宿主，将含有鞭毛蛋白基因ｆｌｉＣｉ的质粒ｐＧＩ４０１５ＢＳ转化ＬＢ５０００，用沙门氏菌普遍转导噬菌体Ｐ２２裂解阳性转化菌，再以此Ｐ２２裂解液转导鞭毛蛋白基因缺陷的减毒都柏林沙门氏菌ＳＬ５９２８，经Ａｍｐ＋和动力双重筛选，获得ｐＧＩ４０１５ＢＳＳＬ５９２８阳性转导子。动力和动力抑制试验、电镜观察和血清学鉴定结果，证实ｆｌｉＣｉ在转导菌中获得了功能性的高效表达。经与大肠杆菌ＬＣ－２ａ（ｈａｇ－，ｒｅｃＡ－）表达ｆｌｉＣｉ结果比较，本研究构建的表达系统优于前者，这为开展以鞭毛蛋白基因为载体表达外源抗原基因的研究奠定了基础，同时为分析鞭毛蛋白免疫分子生物学特性提供了条件。

The expression system for Salmonella gene fliC i was established. The recombinant plasmid pGI4015BS carrying the gene fliC i was used to transform Salmonella typhimurium LB5000 (a restriction negative, modification proficient, non flagellated strain with mutation flaA66) competent cells and then transferred to a flagellin negative, aromatic dependent live vaccine strain of S.dublin SL5928 by transducing using phage P22 HT int, with selection for ampicillin resistance in each case. The ampicillin resistant transductants obtained had functional flagella, as shown both by rapid spreading growth in semisolid medium and by normal assembly of peritrichous flagella investigated under electron microscope. Antigen H1 i expressed in the SL5928 and E.coli Lc 2a hag - variant strain were identified by motility inhibition test with polyclonal rabbit anti H1 i serum. It was further verified that the polyclonal rabbit anti H1 i serum could immobilize and rapidly agglutinate bacteria of strain SL5928 transformed by pGI4015BS in contrast to the not clear agglutination between the serum and bacteria of Lc 2a transformed with same gene. These results showed that this expression system should be a high level one for Salmonclla gene fliC i, and suggested that it would be very useful in the construction of fliC vector for insertion of foreign genes, and the molecular analysis of epitopes on flagellins.