淋病奈瑟菌PIB抗原表位合成肽的研究及PIII蛋白载体的构建

发布时间：2019-06-22 09:29

【摘要】： 淋病奈瑟菌(Neisseria gonorrhoeae),是淋病的病原体。淋病是当今国内外发病率最高的性传播疾病(Sexual Transmitted Disease, STD)之一。人类是淋病奈瑟菌唯一的自然宿主,主要通过性接触而传播。感染淋病奈瑟菌之后,患者发生泌尿生殖系统的损害,表现为尿道炎和宫颈炎,以及附睾炎、盆腔炎等并发症。女性患者还可由于并发症造成不育、宫外孕等严重后果。淋病的诊断主要依据临床表现和实验室检查,而后者是诊断淋病的重要依据。目前常用的实验室检测淋病奈瑟菌的方法是分泌物直接涂片镜检和培养法。但慢性患者或女性病人症状不明显、不典型,涂片的阳性率不高,须依赖于淋病奈瑟菌培养确诊。由于淋病奈瑟菌不易培养,需特殊培养基方能方能培养成功。标本运送使淋病奈瑟菌活力下降也可能造成错误鉴定或漏检。长期以来,许多学者努力寻找一种简单、直接、快速的检测淋病奈瑟菌的方法。近年来,国内外也有将核酸探针技术、PCR方法和连接酶链反应(LCR)应用于淋病奈瑟菌的检测,但这些方法要求设备条件高,且因基层医院目前尚无很好的质量控制和保证,不能作为淋病常规诊断依据,应用受到较大限制。免疫学检测方法,如:酶联免疫法、免疫荧光法和多克隆抗体协同凝集法等也逐渐被运用于淋病奈瑟的检测,并有望成为一种经济实惠、易掌握、易操作的新型淋病奈瑟菌检测手段。而这些方法依赖于淋病奈瑟菌有效抗原的筛选。淋病奈瑟菌的主要抗原成分有菌毛、脂多糖和外膜蛋白。菌毛具有较高的变异性,而脂多糖除有较高变异性外,还有毒性和与宿主碳水化合物有部分相似性,这些因素使它们不能成为制备淋病奈瑟菌检测试剂的成份。外膜蛋白包括PI、PII、和PIII。PI为主要外膜蛋白,占淋病奈瑟菌外膜总重量的60%以上,具有较高的稳定性和免疫原性。PI又可分为两个主要型别,PIA和PIB。表达PIA的淋病奈瑟菌多见于非生殖道的感染。Butt和Fletcher等人对PIB做长期、深入地研究后发现,单克隆抗体SM198能与所有参与测试并且表达PIB的淋病奈瑟菌菌株发生反应。SM198所能识别的抗原表位(epitope)位于PIB上,其中有五个连续的氨基酸呈现出较高的保守性,该序列被定义为SM198识别的最小表位。尽管结构上的变异使PI产生血清型特异性,但是保守的抗原表位给抗体的识别与反应提供了潜在的目标。PIII也是淋病奈瑟菌的外膜抗原之一,稳定地表达于所有淋病奈瑟菌外膜表面。与PI、PII和菌毛不一样,PIII表现出非常高的保守性,所有的淋病奈瑟菌都只表达同一型别的PIII。国内外对PIII的研究报道并不多,这可能与PIII失去成为淋病疫苗研究的候选分子有关,因为机体针对PIII产生的抗体能抑制其它抗体和补体对淋病奈瑟菌的杀菌作用。但是PIII的高度保守性可以被利用来研制淋病奈瑟菌的检测试剂。本研究利用人工合成肽技术合成一段多肽LD1,其序列对应淋病奈瑟菌PIB上的抗原表位所在区域,并用化学方法与钥孔血蓝蛋白(keyhole limpet hemocyanin,KLH)偶联,获得完全抗原LD1-KLH。用该完全抗原免疫动物,以研究其免疫原性;制备抗LD1-KLH多克隆抗血清,检测该抗血清的特异性,为将其用于临床快速诊断研制相关检测试剂奠定实验基础。另外,正确构建淋病奈瑟菌PIII蛋白原核表达质粒,为进一步获得免疫血清作准备,以期制备复合检测试剂。本研究工作分以下二个部分: 一、淋病奈瑟菌外膜蛋白PIB抗原表位合成肽的研究 1.淋病奈瑟菌PIB抗原表位氨基酸序列分析及比对使用DNAMAN对来自于GenBank的18株淋病奈瑟菌PIB氨基酸序列进行对比后发现,抗原表位所在区域中有五个连续的氨基酸序列与参与比对的PIB同区域序列的同源性为100%。BLAST将淋病奈瑟菌参考株R10抗原表位所在区域(15个氨基酸)序列与GenBank里的所有淋病奈瑟菌PIB同区域序列进行比对,同源性在80%以上。以R10抗原表位所在区的这个15个氨基酸作为LD1的序列。 2.抗LD1-KLH多克隆抗体的制备化学合成LD1序列,并与KLH进行化学偶联形成完全抗原LD1-KLH。取200ug/ml和100ug/ml的LD1-KLH完全抗原0.5ml与等体积完全福氏佐剂(CFA)混合,初次免疫家兔和豚鼠;并以同样浓度的LD1-KLH混合等体积不完全福氏佐剂(IFA),在第15、30和45天加强免疫。第4次免疫后10天,心脏取血,收集血清, -20°C保存备用。两种动物均在完全抗原免疫前由耳静脉取血收集非免疫血清作为对照。 3.抗LD1-KLH多克隆抗体的特异性检测 (1)酶联免疫吸附试验(ELISA):ELISA方法检测免疫血清与完全抗原LD1-KLH的反应效价。以25ug/ml的LD1-KLH为抗原,每孔100ul进行包被。动物免疫血清按倍比进行稀释。ELISA检测结果表明,随着免疫血清稀释倍数的增加,反应孔吸光度(A)值逐渐降低,兔抗血清最高效价在1:3200~1:6400之间,豚鼠抗血清最高效价在1:25 600~1:51 200之间。抗LD1-KLH多克隆抗体与LD1-KLH抗原间具有良好的特异性免疫反应。 (2) Western blot:淋病奈瑟菌直接用加样缓冲液裂解,经12%的SDS-聚丙稀酰胺(SDS-PAGE)凝胶电泳分离后,转印到ECL膜上,以Western blot对免疫血清进行检测分析。淋病奈瑟菌裂解物与低分子量标准蛋白Marker一同通过SDS-PAGE分析,以标准蛋白质的相对迁移率为x,标准蛋白质的分子量为y,求回归方程y=f(x),计算显色条带的分子量。结果显示兔抗血清和豚鼠抗血清均与淋病奈瑟菌裂解物有明显的反应条带,而非免疫血清则无明显反应。细菌裂解物经过SDS-PAGE分离再经考马斯亮蓝染色后显示出多条蓝色条带,其中一条的位置对应于Western blot的反应条带,根据回归方程求得其分子量为37 300。 (3)抗LD1-KLH多克隆抗体交叉反应:用ELISA法检测兔抗血清和豚鼠抗血清与淋病奈瑟菌、金黄色葡萄球菌、甲型溶血性链球菌、乙型溶血性链球菌、肺炎链球菌、大肠杆菌和白色念珠菌等不同微生物之间的交叉反应。结果显示无论是兔免疫血清还是豚鼠免疫血清均能与淋病奈瑟菌反应,而与其它参与检测的微生物之间无交叉反应。二、淋病奈瑟菌原核表达载体pGEX-PIII的构建和鉴定常规酚-氯仿法提取淋病奈瑟菌基因组DNA,根据淋病奈瑟菌外膜蛋白PIII基因序列设计一对携带限制性核酸内切酶(BamH I, EcoR I)位点的引物,采用PCR方法获得PIII全基因片段,产物经酚:氯仿:异戊醇抽提后以乙醇沉淀纯化;纯化产物和载体质粒pGEX-3X分别经BamH I,EcoR I酶切后行凝胶电泳回收目的片段和质粒大片段,经T4连接酶连接;连接产物转化到感受态细胞中,经氨苄青霉素抗性、双酶切和测序筛选出阳性克隆,命名为pGEX-PIII。结论:将淋病奈瑟菌外膜蛋白PIB抗原表位和外膜蛋白PIII联合应用于淋病检测试剂的研究在国内未见报导。本研究利用人工合成多肽制备针对淋病奈瑟菌特异性多克隆抗体,并用ELISA和Western blot检测其特异性。结果表明人工合成多肽具有免疫原性,能诱导机体产生识别淋病奈瑟菌抗原表位的特异性抗体。PIII的氨基酸残基数是PIB抗原表位的数十倍,用人工合成的方法来获得将大大增加研究或生产成本。因此,本研究用分子生物学技术成功构建淋病奈瑟菌外膜蛋白PIII原核表达载体pGEX-PIII,为进一步获得免疫血清作准备。总之,本研究的工作为新型淋病奈瑟菌快速诊断试剂的研制提供实验数据。
[Abstract]:Neisseria gonorrhoeae, a pathogen of gonorrhea. Gonorrhea is one of the highest incidence of sexually transmitted diseases (STD) at home and abroad. Human is the only natural host of Neisseria gonorrhoeae, mainly through sexual contact. After the infection of Neisseria gonorrhoeae, the patient has the damage of the genitourinary system, such as the urethritis and cervicitis, and the complications such as epididymitis and pelvic inflammation. The female patient can also cause serious consequences such as infertility, ectopic pregnancy, etc. due to the complications. The diagnosis of gonorrhea is based on the clinical and laboratory tests, and the latter is an important basis for the diagnosis of gonorrhea. The most commonly used methods for the detection of Neisseria gonorrhoeae are direct smear microscopy and tissue culture The symptoms of chronic or female patients are not obvious, and the positive rate of the smear is not high, and it is necessary to rely on the culture of Neisseria gonorrhoeae. It is confirmed that the Neisseria gonorrhoeae is not easy to be cultured, and the special culture medium can only be used for culturing Success. The loss of Neisseria gonorrhoeae may also result in an incorrect identification of the specimen delivery. For a long time, many scholars have tried to find a simple, direct and rapid detection of Neisseria gonorrhoeae. Methods: In recent years, the nucleic acid probe technique, the PCR method and the ligase chain reaction (LCR) have been applied to the detection of Neisseria gonorrhoeae, but these methods require high equipment conditions and can not be used as a routine diagnosis of gonorrhea due to the lack of good quality control and guarantee at the grass-root hospital. Based on, the application is relatively large The method of immunological detection, such as the enzyme-linked immunosorbent assay, the immunofluorescence method and the multi-clone antibody synergistic agglutination method, is also gradually applied to the detection of Neisseria gonorrhoeae, and is expected to be a novel Neisseria gonorrhoeae detection which is economical, easy to master and easy to operate. Means. These methods are dependent on the effective antigen of Neisseria gonorrhoeae. screening. The main antigenic components of Neisseria gonorrhoeae are pili, lipopolysaccharides, And the lipopolysaccharides have partial similarity with the host carbohydrate, so that they can not be used as the detection of Neisseria gonorrhoeae. The outer membrane protein comprises PI, PII, and PIII. The PI is the main outer membrane protein, which accounts for more than 60 percent of the total weight of the outer membrane of the Neisseria gonorrhoeae, and has high stability. and the PI can be divided into two main types, A and PIB. Neisseria gonorrhoeae expressing PIA is found in non-raw materials. A long-term, in-depth study of the PIB by But and Fletcher et al. found that the monoclonal antibody SM198 is capable of interacting with all Neisseria gonorrhoeae participating in the test and expressing the PIB The strain is reacted. The epitope (epitope), which can be recognized by the SM198, is located on the PIB, with five consecutive amino acids exhibiting high conservation, which is defined as the SM198 identification In spite of the structural variation, the PI results in serotype-specific, but the conserved epitope of the antigen provides the identification and reaction of the antibody The potential target. PIII is also one of the outer membrane antigens of Neisseria gonorrhoeae and is stably expressed in all Neisseria gonorrhoeae. The surface of the outer membrane of the bacteria is different from that of PI, PII and pili. PIII shows very high conservation, and all Neisseria gonorrhoeae express only the same type. PIII. There are not many studies on PIII at home and abroad, which may be related to the loss of PIII as a candidate for the study of gonorrhea, since the antibody produced by the body against PIII can inhibit other antibodies and complement to Neisseria gonorrhoeae. But the highly conserved PIII can be used to develop Neisseria gonorrhoeae. The present study uses synthetic peptide to synthesize a segment of polypeptide LD1, the sequence of which corresponds to the region of the antigen epitope on the Neisseria gonorrhoeae PIB and is coupled with the keyhole limpet hemocyanin (KLH) by a chemical method to obtain a complete anti-virus. Original LD1-KLH. Use this complete antigen to immunize animals to study its immunogenicity; prepare anti-LD1-KLH polyclonal antisera, detect the specificity of the antisera, and provide them for clinical and rapid diagnosis and development in addition, the prokaryotic expression plasmid of the Neisseria gonorrhoeae PIII protein is correctly constructed, and the preparation of the immune serum is further obtained, The composite detection reagent is prepared. The research work is divided into two parts: one, the outer membrane of Neisseria gonorrhoeae Study of the synthetic peptide of protein PIB antigen epitope 1. Neisseria gonorrhoeae Analysis of the amino acid sequence of the PIB antigen epitope and the comparison of 18 strains from the GenBank using the DNA MAN in comparison with that amino acid sequence of Neisseria gonorrhoeae, it was found that there were five consecutive amino acid sequence in the region of the antigen epitope and 100% homology with the region sequence of the PIB participating in the comparison. BLAST showed the sequence of the region (15 amino acid) of the region (15 amino acid) of the region (15 amino acid) of the reference strain of Neisseria gonorrhoeae to the GenBank Neisseria gonorrhoeae PI And B is compared with the region sequence, and the homology is more than 80 percent. This 15 amino acid in the region is used as the LD1. Sequence.2. Preparation of LD1-KLH Polyclonal Antibody Synthesis of LD1 Column and chemical coupling with KLH to form complete antigen LD1-KLH.200 ug/ ml and 100 ug/ ml of LD1-KLH complete antigen 0.5 ml mixed with equal volume of complete Freund's adjuvant (CFA), primary immune rabbit and guinea pig, and mixed with the same concentration of LD1-KLH F. F. Freund's adjuvant (IFA), toddler dose on Days 15,30 and 45. 4th 10 days after immunization, blood was taken from the heart, serum was collected, and the serum was stored at -20 掳 C. Both animals The non-immune blood was collected from the ear vein prior to complete antigen immunization. 3. Specific detection of the anti-LD1-KLH polyclonal antibody (1) Enzyme-linked immunosorbent assay (ELISA ): The ELISA method is used to detect the reaction titer of the immune serum and the complete antigen LD1-KLH. .25 ug/ ml of LD1- KLH is an antigen and is coated with 100ul of each well. The animal immune serum is diluted according to the ratio. The results of the ELISA test show that with the increase of the dilution factor of the immune serum, the absorbance of the reaction cell (A) is gradually reduced, and the highest titer of the rabbit antiserum is 1:3200-1:64. Between 00 and 00, the highest titer of the guinea pig antiserum was between 1:25 600 and 1:51 200. The anti-LD1 -KLH polyclonal antibody and LD1-KLH antigen have a good specific immune response. (2) Western blot: Neisseria gonorrhoeae is directly cracked by sample loading buffer, and is separated by SDS-PAGE gel electrophoresis with 12% SDS-PAGE After leaving, the ECL film was transferred to the ECL membrane and the immune serum was analyzed by Western blot. The Neisseria gonorrhoeae lysates were analyzed with the low molecular weight standard protein Marker by SDS-PAGE and the relative mobility of the standard protein was x. And the molecular weight of the standard protein is y, the regression equation y = f (x) is obtained, and the molecular weight of the color developing strip is calculated. The anti-sera of the mice had a clear reaction band with the lysate of Neisseria gonorrhoeae, and the non-immune serum did not react significantly. The bacterial lysates were separated by SDS-PAGE and stained with Coomassie brilliant blue to show a plurality of blue bands, one of which corresponds to the position. The cross-reaction of the anti-LD1-KLH polyclonal antibody with the molecular weight of 37 300 (3) was determined according to the regression equation. The rabbit antiserum and the antiserum of the guinea pig were detected by ELISA. and Neisseria gonorrhoeae, S. aureus, and S. A. haemolyticus, Cross-reaction between different microorganisms such as B. haemolyticus, S. pneumoniae, E. coli and Candida albicans. The results are shown to be either rabbit immune serum or rabbit immune serum. Or the guinea pig's immune serum can be reacted with Neisseria gonorrhoeae, and can be detected by other participants. Non-cross-reaction between microorganisms. Construction of the prokaryotic expression vector pGEX-PIII of Neisseria gonorrhoeae and identification of the genomic DNA of Neisseria gonorrhoeae by conventional phenol-chloroform method, according to Neisseria gonorrhoeae The outer membrane protein PIII gene sequence is designed with a pair of primers carrying the restriction endonuclease (BamH I, EcoR I) site, the PIII whole gene fragment is obtained by the PCR method, the product is purified by ethanol precipitation after the extraction of the isoamyl alcohol, and the purified product and the vector plasmid pGEX-3 and X is respectively digested by BamH I and EcoR I, and the target fragment and a large fragment of the plasmid are recovered by gel electrophoresis, and the target fragment and the plasmid large section are connected through T4 ligase; and the connecting product is turned In the competent cells, the positive clones were screened by the resistance of aminophenylpenicillin, double-enzyme digestion and sequencing, and named pGEX-PIII. Conclusion: The application of the outer membrane protein PIB antigen epitope and the outer membrane protein PIII of the Neisseria gonorrhoeae to the gonorrhea detection reagent is not reported in the country. The polyclonal antibody specific to Neisseria gonorrhoeae is prepared by the synthetic polypeptide, and the antibody is detected by ELISA and Western blot. The result shows that the synthetic polypeptide has the immunogenicity and can induce the organism to produce the specific antibody recognizing the epitope of the Neisseria gonorrhoeae. The amino acid residue base of PIII is dozens of times of the epitope of the PIB antigen, and the research or production cost can be greatly increased by using the synthetic method. Therefore, the molecular biology technique for the research Successful construction of the prokaryotic expression vector pGEX-PIII of Neisseria gonorrhoeae outer membrane protein PIII
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R759.2;R392

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