关键词： 钩体；重组DNA；表达蛋白p68；抗原性；免疫保护

摘要目的寻找钩体病基因工程疫苗保护性抗原候选。方法(1)鸟枪法构建赖型钩体017株基因库；(2)α互补、DNA 原位杂交筛选重组质粒；(3)Southern 杂交行 DNA 同源性分析；(4)双脱氧链中止测重组 DNA 序列及与外膜蛋白基因(omp)匹配；(5)IPTG 诱导重组子表达；(6)免疫印迹、MAT、EIA、MTT检测表达蛋白抗原特性及IL—2、IL—6活性；(7)纯化表达蛋白p68进行豚鼠主动免疫/攻击实验，观免疫保护性。结果(1)重组质粒 pDJH₂(亚克隆pDJt)插入片段1.9kb,与各致病钩体不同片段 DNA 高度同源；(2)序列测定插入片段实际1811bp,推测有2个读框(ORF)，各有启动子、终止密码。查 Gen Bank/EMBL 无类似序列；(3)表达蛋白分子量68kD(p68)；(4)p68是胸腺依赖性(TD)抗原，有良好抗原特性，抗血清效价1：524288，(EIA)具很强的动物免疫保护作用。结论(1)p68编码基因是致病钩体保护性抗原基因；(2)p68是致病钩体外膜保护性抗原，可作钩体基因疫苗候选。

STUDY OF RECOMBINANT DNA AND P68,AN ANTIGEN EXPRESSED FROM THE RECOMBINANT DNA OF LEPTOSPIRA INTERROGANS SEROVAR LAI

Jiang NanDai BaominLi ShenfuFang Zhimao

(Department of Infection Disease,People's Hospital of Sichuan Province,Chengdu,610071)

ABSTRACTAim To look for a antigen candidate of gene engineering vaccine for leptospirosis.Characteristic of p68,a protective antigen expressed from recombinant gene of L.interrogans serovar Lai strain 017 was studied.Methods(1)random cloning was used to construction a gene library of L.interrogans serovar Lai strain 017;(2)Situ blotting was used to selection recombinant plasmid in the bank;(3)Southern blotting was seleted to research on the DNA homlogy;(4)Sanger double-dideoxy-mediated chaintermination was employed to check the sequence of the fragment insered and its alignment with other bactereal omp(outer membrane protein);(5)To induce the recombinant protein expressed,IPTG was adoped;(6)Antigenicity of the protein expressed was studied with immunoblotting.EIA and MAT;(7)MTT was taken to test the actions of IL-2 and IL-6 in guinea pigs immunized with the protein expressed;(8)Initiative immunization was carried out to identify the immuno/protection on the model of guinea pigs with the protein expressed.Results(1)A recombinant clone,designated as pDJH₂ (subclone was pDJt)in the bank was obtained.It contained 1.9kb DNA fragment from L.Strain 017;(2)The 1.9kb had highly homlogy with pathogenic leptospires;(3)The sequence analysis showed that total numbers of nt for the inserted ORA were 1811bp.There were two open reading frames(ORF)of 565 nt and 662nt there are promoters,terminators and initiatin codons respectively,in ORFs.There were not similar sequence in the GenBank/EMBL;(4)The molecular weight of protein expressed of pDJH₂/pDJt was 68kD(p68);(5)p68 could elicit a strong and specific humoral immune response that T and B cell took part in.Antiserum titer of p68 was 1:524288(guinea pigs,EIA).The activity of IL-6 was 83.25 IU*ml,IL-2 was 75IU*ml,and p68 was idetified as a major antigen of OMP from L.serovarLai;(6)Guinea pigs immunized with p68 could resist well challenge of the strong toxicity L.serovar Lai strain 017 .Conclusion(1)Code gene of p68 was a protective antigen of pathogenic leptospires;(2)p68 was a good protective antigen for leptospirsis and candidate for gene engineering vaccine of leptospirosis.

KEY WORKSLeptospira Recombinant DNA Protein(p68) Exrpessed Antigenicity Immuno/Protection

钩端螺旋体病(简称钩体病)是世界范围人兽共患病。强毒力赖型钩体017株可引起致命肺弥漫出血(PDH)，严重威胁人民健康。因此，钩体病预防实属必要。然致病钩体保护性抗原基因的寻找并非件易事，这无疑给钩体基因疫苗研究带来阻力。基于此，作者构建了问号钩体赖型017株基因库，筛选出重组质粒 pDJH₂(亚克隆pDJt)。对插入片段(1.9kb)进行了 DNA 分析；对插入基因表达蛋白p68也进行了研究，现报道如下。

1材料和方法

1.1材料钩端螺旋体成都生物制品研究所，北京生物制品鉴定所提供，华西医科大学钩体研究室保存，Korthorf 培养基28℃培养7天，达1×10⁷条/ml,定期感染豚鼠保存毒力。

质粒：pUC18(四川联大张义正教授惠赠)，pT7—7(美国波斯顿哈佛医学院 Stanley Tabor 教授惠赠)。

宿主菌：大肠杆菌(E.coli)JM109(DE₃)(Promega),E.coli JM103(四川联合大学张义正教授惠赠)。

主要试剂：均为 Sigma,Promega,Boehringer Mannheim,GIBco 等或进口分装，国产优质纯，分析纯产品。

1.2方法

1.2.1钩体017株基因库构建方法参见文献^［1，2］。

1.2.2重组克隆筛选(1)α互补，DNA 原位杂交(探针是017株钩体全钩DNA)，EcoRI 酶切筛选重组质粒：方法参照文献^［2］。(2)重组质粒插入片段与各致病钩体 DNA 同源性分析(Southern blotting)：探针1是1.9kb 017株钩体重组 DNA 片段；探针2是L.alstoni 2.5kb DNA 片段(含ompL₁全结构基因)。方法按地高辛标记检测药盒说明。

1.2.3重组子亚克隆及插入片段 DNA 测序亚克隆的目的片段是1.9kb，载体pT7—7，宿主菌 E.coli JM109(DE₃)。1.9kb 插入片段 DNA 测序及与其它 omp 同源匹配，方法见文献^［2］。

1.2.4重组子表达26℃IPTG 诱导重组质粒(克隆/亚克隆)表达，蛋白酶K常规消化定性^［2］。

1.2.5P68 抗原特性研究(1)斑点酶联免疫吸附试验(Dot-EIA)：包被抗原：p68，一抗：017株钩体全钩抗血清，效价1∶10000。工作浓度1∶200，二抗HRP标记，工作浓度1∶1000。方法按药盒说明。(2)免疫印迹(Immunoblotting)：抗原p68 SDS-PAGE，一抗：017株钩体外膜蛋白(OMP)抗血清，效价1∶12800，工作浓度 1∶100，二抗HRP 标记，工作浓度0.5u/ml,方法按常规。

1.2.6p68 免疫/保护豚鼠实验健康豚鼠(150g-180g)随机分4组，14只/组，免疫原：p68,pDJt,PT7-7(阴性对照)，WCV(阳性对照)。免疫剂量：p68 50μg/次/只，余均为1ml菌液/次/只。(1×10⁸/ml菌，超声击碎)免疫途径：多点皮下注射+双腿肌肉注射。初免加等量完全弗氏佐剂。第一次加强在初免后14天，加等量不完全弗氏佐剂。第二次加强在初免后24天，不加佐剂，抗原量加倍，攻击于第二次加强后10天进行。攻击量为1ml(5×10⁸/ml)强毒力活017株钩体。1/2腹部皮下注射，1/2腹腔注射。观察指标：死亡率，攻击后10天处死存活动物观肺出血情况。

2结果

2.1赖型钩体017株重组 DNA 研究经α互补，DNA 杂交(017株钩体全钩 DNA 为探针)，EcoRI 酶切筛选出重组质粒pDJH₂，插入片段1.9kb，与各致病钩体 DNA 不同片段高度同源，与 L.alstoni 2.5kb DNA 探针高度同源；pDJH₂ 亚克隆重组子pDJt 经 EcoRI、Bgl Ⅱ双酶消化电泳示1.9kb片段正向插入(上述结果未列出)，1.9kb DNA 序列测定结果见文献^［3］。重组质粒构建程序见图1。

图1重组质粒构建程序

Fig1.Scheme of construction of recombinant plasmid (containing 1.9kb fragment inserted of L.interrogans serovar Lai strain017)

2.2重组质粒pDJH₂/pDJt表达SDS—PAGE见pDJH₂/pDJt 68kD 处有新带，与017 株钩体 OMP 相应带位置一致。阴性对照pT7-7+JM109(DE₃）68kD 处未见新带(图2)；68kD 带经蛋白酶K消化后消失。

图2.pDJH₂/pDJt SDS-PAGE(IPTG 诱导表达)

Fig2.SDS-PAGE of protein expressed of pDJH₂/pDJt,induced with IPTG (The new band indicated by the arrows)

1.Marker of protein;2.OMP of L.interrogans Serovar Lai Strain 017;(positive controlling);3.6.pDJt;4.pT7-7(neg