PROTECTION AGAINST LEPTOSPIROSIS BY IMMU NIZATION WITH PLASMID DNA ENCODING 33 kDa ENDOFLAGELLIN OF L.INTERROGANS SEROVAR LAI

To evaluate how the efficacy of DNA inocutation affects the ability to raise protective immunity against Leptospira. [WT5”BX]Methods.[WT5”BZ] A pair of oligonucleotide primers were designed to amplify the endoflagellar gene of L. interrogans sensu stricto serovar lai. An approximately 840bp fragment was generated with PCR and inserted into VR1012, a plasmid DNA expression vector, after the fragment and VR1012 were digested respectively with EcoRV and Sal I. A recombinant plasmid designated as VR1012+flaB2 was obtained. The vector, VR1012 consits of a pUC18 backbone with the cytomegalovirus(CMV) IE1 enhancer, promoter, and intron A, transcription regulatory elements and the BGH polyadenylation sequences driving the expressing of leptospiral endoflagellar gene of L. interrogans sensu stricto serovar lai. Plasmid encoding leptospiral endoflagellin gene was injected into quadriceps of NZW rabbits. [WT5”BX]Results.[WT5”BZ]This resulted in the generation of specific leptospiral antibody with high ELISA titer (1:32768) in the rabbits. Immuno/protection was performed in guinea pigs without adjuvant. The group“VR1012+flaB2” showed higher survival rate(90%,9/10 animals),compared with the group “VR1012 lack flaB2” and the group “normal saline”. [WT5”BX]Conclusion.[WT5”BZ]The technique of DNA vaccine has potential advantages over certain other vaccine preparation technologies. However whether DNA vaccine will be useful for vaccine development remains to be tested.

赖型钩体flaB2与VR1012中的CpG基序分析

目的 对问号赖型钩端螺旋体 (赖型钩体 ) DNA疫苗〔包括内鞭毛蛋白基因 (fla B2 )和质粒 DNA表达载体 (VR10 12 )〕的 Cp G基序 (Cp G motifs)进行分析 ,为 DNA疫苗免疫机制的阐明和提高 DNA疫苗的效能奠定基础。方法 以 fla B2与 VR10 12构建重组 DNA的免疫原 ,对 fla B2及 VR10 12全核苷酸序列进行计算机分析 (分类、计数和定位 )。结果 Cp G的“C”的侧翼为两个嘌呤 ,“G”的侧翼为两个嘧啶 ,在 fla B2中共 3个 ,分别为GACGCT,GACGTC和 GACGCC;在 VR10 12中共 11个 ,分别为 GACGTC1个 ,GACGCT2个 ,GACGCC1个 ,GACGTT1个 ,GGCGTT2个 ,GGCGCT2个 ,GGCGCC1个 ,AACGCT1个 ,其中特别重要的 TGACGTCA4个和 TAACGCCA有 1个 ,位于 5′端 4 5 6～ 4 6 3;5 0 9～ 5 16 ;5 92～ 5 99;778～ 785和 4 86～ 4 93;4个 TGACGTCA和 1个TAACGCCA均位于 5′端且相对集中。结论 赖型钩体 fla B2与 VR10 12构成的 DNA疫苗含有 TGACGTCA等Cp G,这些基序又称免疫刺激序列 ,构成了

问号赖型钩体与七日热型钩体内鞭毛蛋白基因的克隆及序列分析

目的 筛选钩体核酸疫苗的候选株 ,并从基因水平解释钩体血清型的特异性。方法 通过 PCR方法扩增赖型 0 17株及七日热型 5 6 6 10株钩体的内鞭毛蛋白 (fla B)基因 ;经 T/A快速克隆法将 PCR产物连接于PGEM- T Easy Vector载体。用 α-互补法、PCR法及酶切分析鉴定重组质粒 ,获得了重组质粒 p DHTF0 17和p DHTF6 10 ;双脱氧终止法对两个重组质粒中的克隆化 fla B基因进行测序。结果 两型 fla B基因长 85 2 bp,编码2 83个氨基酸。两个基因的限制性内切酶位点相似 ,含多个常见酶切位点。基因编码的蛋白质预测相对分子质量为31.3× 10 3。结论 几个血清型的 fla B基因同源性达 90 %～ 99%。

PCR AMPLIFICATION, MOLECULAR CLONING,DNA SEQUENCE ANALYSIS AND IMMUNO/PROTECTION IN BALB/C MICE OF THE 33 kDa ENDOFLAGELLAR PROTEIN OF L.INTERRORGANS SEROVAR LAI

A pair of oligonucleotide primers were designed to amplify the endoflagella gene of L. Interrogans serovar lai. An approximately 840 bp fragment was generated with PCR and inserted into plasmid pUC8,after the fragment and pUC8 were digested respectively with BamHI and PstI. A recombinant plasmid (designated as pLF1 ) was obtained. SDS-PAGE analysis indicated that a 33 kDa was expressed in E. Coli JM103 harboring pLF1 and the expression level of the protein was 11 % of the total bacterial soluble proteins. Western blot analysis showed that the protein band could be recognized by the antiserum against the endoflegella (Axial filament ) of Leptospira interrt,gans serover lai. Nucleotide sequence data showed an open reading frame encoding 282 aminoacids residues,corresponding to a protein of molecular weight 33. 6kDa. Comparison of the deduced endoflagellar subunit protein (flaB) amino acid sequence with flagellins from other bacteria revealed a high level of identity with the Trehoema pallid um flaB proteins. Immunization/protection experiment was performed on the model of BALB/C mice and showed that there was higher survival rate in the group JM103-pLE1 than in the group JM103-pUC8.

问号赖型钩体内鞭毛基因重组质粒的构建及其在大肠杆菌的表达

为了获得重组表达的钩体内鞭毛亚单位蛋白抗原，以探讨制备新疫苗的途径，利用聚合酶链反应扩增其编码基因完整的开放阅读框架，扩增产物经ＳａｌⅠ、ＣｌａⅠ双酶切消化后定向克隆于ｐＴ７－７表达载体上。重组质粒转化入大肠杆菌ＪＭ１０９（ＤＥ３）中，经ＩＰＴＧ诱导表达，ＳＤＳ－ＰＡＧＥ显示此产物相对分子量为３４ｋｄ，产量占细菌总蛋白的１１．８％。用插入片段为探针，Ｓｏｕｔｈｅｒｎ杂交提示可能有两个相关的基因存在于钩体的基因组内。Ｗｅｓｔｅｒｎ杂交证实该表达蛋白可与内鞭毛抗血清在３４ｋｄ处出现印迹，与钩体内鞭毛同类蛋白带位置一致，本实验克隆了钩体内鞭毛Ｂ类基因并表达了相应抗原。