【作者】 文华；

【导师】 裴晓方；

【作者基本信息】 四川大学 ， 营养与食品卫生， 2004， 硕士

【摘要】 目的: 扩增淋球菌外膜蛋白PI抗原基因,构建pBS-T-PI NG克隆重组子,测定分析PI序列;构建pET30b-PI-NG重组子,在大肠杆菌中诱导表达淋球菌外膜蛋白PI,为后续PI蛋白免疫学特性的研究、抗体制备、以及预防淋病疫苗的研制奠定基础。 方法: 本研究分为三部分:第一部分是淋球菌基因组的提取及外膜蛋白PI基因的PCR扩增。提取采集到的四个临床淋球菌菌株细菌基因组,针对其外膜蛋白Porin I（PI）基因设计特异性引物,PCR扩增PI基因;第二部分是淋球菌外膜蛋白PI基因的克隆与PI基因的序列分析。将目的基因PI与克隆载体pBS-T在连接酶作用下连接,转化DH5α感受态细胞,抗生素平板、蓝白菌落筛选阳性克隆,质粒抽提,双酶切鉴定,基因测序,与GenBank的相应序列进行序列分析比较;第三部分是淋球菌外膜蛋白PI基因表达质粒重组子的构建与表达。从重组的克隆质粒pBS-T-PI中切取目的基因,插入表达质粒载体pET30b中,转化DH5α感受态细胞,抗生素平板筛选阳性重组子,质粒提取,双酶切和PCR鉴定,选取鉴定正确的阳性重组子转化表达宿主大肠杆菌BL21（DE3）,IPTG诱导蛋白质表达,SDS-PAGE初步分析。结果: 1.四株临床淋球菌（分别命名为NG₁、NG₂、NG₃、NG₄）的基因组经PCR扩增得到0.9kb～1.0kb范围内的预期长度目的片段,分别命名为PI NG₁、PI NG₂、PI NG₃和PI NG₄。 2.将PI NG₁、PI NG₂、PI NG₃和PI NG₄分别插入pBS-T克隆质粒,构建了更多还原

【Abstract】 Object:Porin I （PI） is the major outer membrane protein of Neisseria gonorrhoeae. In this research we described a method by which the gene encoding PI was cloned into an expression plasmid and then PI protein was expressed in E.coil. The expressed PI protein will be applied in the further research for PI antigenicity and immunological activity. This will be very helpful for the further construction of vaccines for prevention of Neisseria gonorrhoeae infection.Methods:1. Genome extraction of Neisseria gonorrhoeae. Four clinic isolates of Neisseria gonorrhoeae （named NG₁, NG₂, NG₃, and NG₄ respectively） were collected, and then the genome of these strains were extracted. The gene encoding for PI of Neisseria gonorrhoeae was amplified by PCR （polymerase chain reaction）, with a pair of particular designed primers.2. Cloning and sequence analysis of Neisseria gonorrhoeae major outer membrane protein PI. The genes encoding for PI were inserted into the cloning vector pBS-T, transformated to E.coli DH5α competent cells. The subsequent clones were demonstrated as ampicillin resistance and white colonies. EcoR I and Xho I were applied to confirm the recombinant plasmids. And sequence analysis was also performed to verify the recombination plasmids and the diversity of the PI gene in four strains which infect Chinese.3. Construction of expression recombinant plasmids pET30b-PI and protein expression. PI gene fragments were purified and cut from pBS-T-PI recombinant plasmids, inserted into expression vector pET30b, transformated into E.coli DH5α competent cells. The target clones were selected by using Kanamycin resistance, and were detected by enzyme digestion and PCR. The conformed recombinant plasmids pET30b-PI was transformated to E.coli. BL21 （DE）, and the the protein expression were induced by add IPTG in the inoculums. The expressed protein was analyzed bySDS-PAGE.Results:1. Four target outer membrane protein PI gene fragments designed as PI NGi x PI NG2x PI NG3 and PI NG4 were obtained by PCR from four clinic isolates of Neisseria gonorrhoeae （named NGi, NG2, NG3, and NG4 respectively）. The lengths of these four fragments were range from 0.9kb to 1 .Okb.2. PI NGi > PI NG2> PI NG3 and PI NG4 were inserted separately into pBS-T vector to construct pBS-T-PI NGh pBS-T-PI NG2, pBS-T-PI NG3 and pBS-T-PI NG4 recombinants. These recombinants were conformed by both restrict enzyme digestion and sequencing.3. According to the sequencing data, the length of PI NGi and PI NG2 were 907bp and encoded a peptide of 302 amino acids. And the length of PI NG3 and PI NG4 were 970bp and encoded a peptide of 323 amino acids.4. In compare with the PI sequences in GenBank, PI NGi had two base differences with that of Neisseria gonorrhoeae 4805 strain （GI: 3925497//ACCESSION: AF090819）. The homologous rate was 99%. PI NG2 had four base differences with that of Neisseria gonorrhoeae 4805 strain. The homologous rate was also 99%. PI NG3 had two base differences with that of Neisseria gonorrhoeae SU106 strain （GI: 1763329//ACCESSION: U75639） with the same 99% homologous rate. But the PI NG4 had a higher homologous rate （98%） with an 18 base differences when compared with that of 4174 strain （GI: 3925461//ACCESSION: AF090801）. When compared within the four PI NGs, the homologous rate of the DNA was 74% with a 232 base differences, and the protein homologous rate was 67% accordingly. PI NGi and PI NG2 were more likely belong to PIA subtype whereas PI NG3 and PI NG4 were more likely sit in PIB subfamily.5. PI NGi x PI NG2> PI NG3 and PI NG4 were inserted separately into pET30b vector to construct pET30b-PI NGi, pET30b-PI NG2, pET30b-PI NG3 and pET30b-PI NG4 recombinants. These recombinants were conformed by both restrict enzyme digestion and PCR.6. pET30b-PI NGi, pET30b-PI NG2, pET30b-PI NG3 and pET30b-PI NG4 recombinants were transformed into BL21（DE3） expression host competent cells and protein expression were induced by adding IPTG. All strains but not pET30b-PI NG2 demonstrated obvious target protein bands on SDS-PAGE gel.Conclusion:1. Four target outer membrane protein PI gene fragments obtained from four clinic isolates of Neisseria gonorrhoeae were cloned successfully into pBS-T vector and four recombinant plasmids designed as pBS-T-PI NGi ^ pBS-T-PI NG2n pBS-T-PI NG3 and pBS-T-PI NG4 were obtained.2. Sequencing analysis on the recombinant pBS-T-PI NGs were made. This is the first sequence report on Neisseria gonorrhoeae outer membrane protein PI gene isolated from infected person in China. According to the DNA and protein sequences, PI NGi and PI NG2 were more likely belong to PI A subtype whereas PI NG3 and PI NG4 were more likely sit in PIB subfamily.3. The expression recombinant plasmids pET30b-PINGi, pET30b-PING2, pET30b-PING3, and pET30b-PING4 were constructed successfully. Target proteins were observed on SDS-PAGE gel. The expressed PI protein will be applied in the further research for PI antigenicity and immunological activity. This will be very helpful for the further construction of preventve vaccines on Neisseria gonorrhoeae infection.更多还原