论文部分内容阅读
为了探讨ｐＤＪＨ２的１．９ｋｂ０１７株钩体ＤＮＡ片段可否作为钩体重组疫苗广谱保护性抗原的候选，采用重组ＤＮＡ技术，将ｐＤＪＨ２的１．９ｋｂＤＮＡ片段分别与ｐＴ７－７，ｐＲＳＥＴ系列重组，转化入大肠杆菌进行亚克隆，ＩＰＴＧ诱导表达。结果显示：阳性亚克隆ｐＤＪｔ．ｐＤＪｒＢ１能在大肠杆菌中高效表达；对其进行ＳＤＳ－ＰＡＧＥ分析，可见６８ｋｄ和２３ｋｄ处出现新带，与钩体０１７株外膜同分子量蛋白带位置一致，免疫印迹（特异性抗０１７株外膜抗血清）在６８ｋｄ和２３ｋｄ处分别有印迹；用ｐＤＪｔ亚克隆重组质粒主动免疫豚鼠，可抵抗强毒力株攻击，表现出免疫保护作用。本实验结果提示：（１）ｐＤＪＨ２１．９ｋｂ重组ＤＮＡ片段能以不同质粒为载体，在不同大肠杆菌株中高效表达；（２）该重组ＤＮＡ片段表达产物——６８ｋｄ和２３ｋｄ蛋白，可能是赖型钩体０１７株外膜的保护性抗原。
In order to investigate whether the 1.9kb017 leptospiral DNA fragment of pDJH2 could be used as a candidate for the broad-spectrum protective antigen of recombinant leptospira vaccine, a 1.9kb DNA fragment of pDJH2 was recombined with pT7-7 and pRSET series respectively using recombinant DNA technology and transformed into E. coli was subcloned and induced by IPTG. The results showed that: positive subclone pDJt. pDJrB1 was highly expressed in Escherichia coli. SDS-PAGE analysis showed that the new band appeared at 68kd and 23kd, which was consistent with the same molecular weight band of 017 outer membrane of Leptospira. Immunoblotting (specific anti-017 outer membrane Antiserum) were imprinted at 68kd and 23kd, respectively. The recombinant plasmid pDJt was subcloned into active guinea pigs to resist the challenge of virulent strains and showed an immunoprotective effect. The results of this experiment suggest that: (1) pDJH21.9kb recombinant DNA fragment can be efficiently expressed in different Escherichia coli strains with different plasmids as the vector; (2) The expression products of the recombinant DNA fragments - 68kd and 23kd may be dependent Leptospira 017 outer membrane protective antigen.