问号赖型钩体重组质粒pDJH_2　DNA　序列测定及其与其它细菌omp基因序列比较分析

Research on the Recombinant Plasmid pDJH 2 of L.interrogans serovar lai:Sequencing and Alignment with Other Known Bacterial Omp Sequence

对ｐＤＪＨ２片段ＤＮＡ进行了序列测定，结果：插入片段为１８１１个核苷酸；可读框２个：ＯＲＦ１，１－５６５ｂｐ；ＯＲＦ２（从最后一个密码倒算）１－６６２ｂｐ，计算机序列同源性或类似性匹配（ａｌｉｇｎｍｅｎｔ）表明，ＯＲＦ１与ＯＲＦ２的核苷酸类似性为４９．３６％；ＯＲＦ１与Ｌ．ｋｉｒｓｃｈｎｅｒｉ（Ｌ．ａｌｓｔｏｎｉ）流感伤寒型ｏｍｐ核苷酸序列类似性为４９．２６％；ＯＲＦ２与Ｌ．ａｌｓｔｏｎｉ类似性为５１．５６％；ＯＲＦ１与其它细菌（包括螺旋体）ｏｍｐ核苷酸序列对准比较，序列类似性均在４３％以上。经查阅ＥＭＢＬＤＮＡ序列数据库未见类似序列。作者认为ｐＤＪＨ２的１．９ｋｂ插入ＤＮＡ片段（ＯＲＦ１与ＯＲＦ２）可能是具有保护作用的ｏｍｐ基因片段。

The Leptospira whole cell vaccine (LWCV) currently used in China is safe and effctive, but the immunity following vaccination with two doses of the fluid medium vaccine is of low order. The duration of immunity conferred by this vaccine is rather short, six months or at most one year. Therefore, it is necessary to develop new generation vaccines against Leptospirosis for the developing world. In this paper we report the sequencing of the insert fragment of pDJH 2 from genomic DNA of L. interrogans sevovar lai strain 017 and its alignment with other bacterial omp sequences. A genomic library of Leptospira interrogaans serovar lai strain 017 was constructed with the plasmid vector pUC18. A recombinant plasmid designated pJDH 2 was screened from the genomic library. Inserted fragment of pDH 2 is 1.9kb by gel electrophoresis. Immunization/protection was studied in BALB/c mice model. The results showed highly significant difference between pDJH 2 and pUC18 (control). Inserted fragment of pDJH 2 DNA sequencing was performed by Dr Yan Zhengxin (Max Planck Institut fur Biology. Tubingen, Germany). Insert fragment was cloned into pBluescript Ⅱ KS + (stratagene) and sequenced by using AB1 (Applied Bio Systems, Model 373A). Two open reading frames of 565 and 662 nucleotides were identified. There were identifiable initiation codons, terminators, Shine Dalgano ribosome combining site, Pribnow boxes and Sextama boxes within the 2 sequenced regions. Nucleotide sequences were analysed using Gene Work, a suit of computer program developed by Department of Biochemistry St. Jude Children′s Research Hospital Memphis. U.S.A. The results of formatted alignment showed the predicted nucleotide sequence of ORF1 of the serovar lai had significant similarity with ORF2(49.36%), L.kirschneri ompL1 (49.26%), Borrelia burgdoferi omp (48.97%), Treponema phagedenis omp (47.3%); Salmonella typhimurium ompC(46.87%), Yersiniaenterocolitica ompH (46.7%), Leptospira borgpeterseni pfap (46.3%), and Serratia marcescens omp (43.3%). The close relationship of the pDJH 2 ORF1 and ORF2 nucleotide sequences from Leptospira kirschneri ompL 1 is apparent. Whether the recombinant pDJH 2 will prove useful for vaccine development remains to be tested.