【摘要】： 淋病奈瑟菌(Neisseria gonorrhoeae),是淋病的病原體。淋病是當(dāng)今國內(nèi)外發(fā)病率最高的性傳播疾病(Sexual Transmitted Disease, STD)之一。人類是淋病奈瑟菌唯一的自然宿主,主要通過性接觸而傳播。感染淋病奈瑟菌之后,患者發(fā)生泌尿生殖系統(tǒng)的損害,表現(xiàn)為尿道炎和宮頸炎,以及附睪炎、盆腔炎等并發(fā)癥。女性患者還可由于并發(fā)癥造成不育、宮外孕等嚴(yán)重后果。淋病的診斷主要依據(jù)臨床表現(xiàn)和實驗室檢查,而后者是診斷淋病的重要依據(jù)。 目前常用的實驗室檢測淋病奈瑟菌的方法是分泌物直接涂片鏡檢和培養(yǎng)法。但慢性患者或女性病人癥狀不明顯、不典型,涂片的陽性率不高,須依賴于淋病奈瑟菌培養(yǎng)確診。由于淋病奈瑟菌不易培養(yǎng),需特殊培養(yǎng)基方能方能培養(yǎng)成功。標(biāo)本運(yùn)送使淋病奈瑟菌活力下降也可能造成錯誤鑒定或漏檢。長期以來,許多學(xué)者努力尋找一種簡單、直接、快速的檢測淋病奈瑟菌的方法。近年來,國內(nèi)外也有將核酸探針技術(shù)、PCR方法和連接酶鏈反應(yīng)(LCR)應(yīng)用于淋病奈瑟菌的檢測,但這些方法要求設(shè)備條件高,且因基層醫(yī)院目前尚無很好的質(zhì)量控制和保證,不能作為淋病常規(guī)診斷依據(jù),應(yīng)用受到較大限制。免疫學(xué)檢測方法,如:酶聯(lián)免疫法、免疫熒光法和多克隆抗體協(xié)同凝集法等也逐漸被運(yùn)用于淋病奈瑟的檢測,并有望成為一種經(jīng)濟(jì)實惠、易掌握、易操作的新型淋病奈瑟菌檢測手段。而這些方法依賴于淋病奈瑟菌有效抗原的篩選。 淋病奈瑟菌的主要抗原成分有菌毛、脂多糖和外膜蛋白。菌毛具有較高的變異性,而脂多糖除有較高變異性外,還有毒性和與宿主碳水化合物有部分相似性,這些因素使它們不能成為制備淋病奈瑟菌檢測試劑的成份。外膜蛋白包括PI、PII、和PIII。PI為主要外膜蛋白,占淋病奈瑟菌外膜總重量的60%以上,具有較高的穩(wěn)定性和免疫原性。PI又可分為兩個主要型別,PIA和PIB。表達(dá)PIA的淋病奈瑟菌多見于非生殖道的感染。Butt和Fletcher等人對PIB做長期、深入地研究后發(fā)現(xiàn),單克隆抗體SM198能與所有參與測試并且表達(dá)PIB的淋病奈瑟菌菌株發(fā)生反應(yīng)。SM198所能識別的抗原表位(epitope)位于PIB上,其中有五個連續(xù)的氨基酸呈現(xiàn)出較高的保守性,該序列被定義為SM198識別的最小表位。盡管結(jié)構(gòu)上的變異使PI產(chǎn)生血清型特異性,但是保守的抗原表位給抗體的識別與反應(yīng)提供了潛在的目標(biāo)。PIII也是淋病奈瑟菌的外膜抗原之一,穩(wěn)定地表達(dá)于所有淋病奈瑟菌外膜表面。與PI、PII和菌毛不一樣,PIII表現(xiàn)出非常高的保守性,所有的淋病奈瑟菌都只表達(dá)同一型別的PIII。國內(nèi)外對PIII的研究報道并不多,這可能與PIII失去成為淋病疫苗研究的候選分子有關(guān),因為機(jī)體針對PIII產(chǎn)生的抗體能抑制其它抗體和補(bǔ)體對淋病奈瑟菌的殺菌作用。但是PIII的高度保守性可以被利用來研制淋病奈瑟菌的檢測試劑。 本研究利用人工合成肽技術(shù)合成一段多肽LD1,其序列對應(yīng)淋病奈瑟菌PIB上的抗原表位所在區(qū)域,并用化學(xué)方法與鑰孔血藍(lán)蛋白(keyhole limpet hemocyanin,KLH)偶聯(lián),獲得完全抗原LD1-KLH。用該完全抗原免疫動物,以研究其免疫原性;制備抗LD1-KLH多克隆抗血清,檢測該抗血清的特異性,為將其用于臨床快速診斷研制相關(guān)檢測試劑奠定實驗基礎(chǔ)。另外,正確構(gòu)建淋病奈瑟菌PIII蛋白原核表達(dá)質(zhì)粒,為進(jìn)一步獲得免疫血清作準(zhǔn)備,以期制備復(fù)合檢測試劑。 本研究工作分以下二個部分: 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對 使用DNAMAN對來自于GenBank的18株淋病奈瑟菌PIB氨基酸序列進(jìn)行對比后發(fā)現(xiàn),抗原表位所在區(qū)域中有五個連續(xù)的氨基酸序列與參與比對的PIB同區(qū)域序列的同源性為100%。BLAST將淋病奈瑟菌參考株R10抗原表位所在區(qū)域(15個氨基酸)序列與GenBank里的所有淋病奈瑟菌PIB同區(qū)域序列進(jìn)行比對,同源性在80%以上。以R10抗原表位所在區(qū)的這個15個氨基酸作為LD1的序列。 2.抗LD1-KLH多克隆抗體的制備 化學(xué)合成LD1序列,并與KLH進(jìn)行化學(xué)偶聯(lián)形成完全抗原LD1-KLH。取200ug/ml和100ug/ml的LD1-KLH完全抗原0.5ml與等體積完全福氏佐劑(CFA)混合,初次免疫家兔和豚鼠;并以同樣濃度的LD1-KLH混合等體積不完全福氏佐劑(IFA),在第15、30和45天加強(qiáng)免疫。第4次免疫后10天,心臟取血,收集血清, -20°C保存?zhèn)溆�。兩種動物均在完全抗原免疫前由耳靜脈取血收集非免疫血清作為對照。 3.抗LD1-KLH多克隆抗體的特異性檢測 (1)酶聯(lián)免疫吸附試驗(ELISA):ELISA方法檢測免疫血清與完全抗原LD1-KLH的反應(yīng)效價。以25ug/ml的LD1-KLH為抗原,每孔100ul進(jìn)行包被。動物免疫血清按倍比進(jìn)行稀釋。ELISA檢測結(jié)果表明,隨著免疫血清稀釋倍數(shù)的增加,反應(yīng)孔吸光度(A)值逐漸降低,兔抗血清最高效價在1:3200~1:6400之間,豚鼠抗血清最高效價在1:25 600~1:51 200之間。抗LD1-KLH多克隆抗體與LD1-KLH抗原間具有良好的特異性免疫反應(yīng)。 (2) Western blot:淋病奈瑟菌直接用加樣緩沖液裂解,經(jīng)12%的SDS-聚丙稀酰胺(SDS-PAGE)凝膠電泳分離后,轉(zhuǎn)印到ECL膜上,以Western blot對免疫血清進(jìn)行檢測分析。淋病奈瑟菌裂解物與低分子量標(biāo)準(zhǔn)蛋白Marker一同通過SDS-PAGE分析,以標(biāo)準(zhǔn)蛋白質(zhì)的相對遷移率為x,標(biāo)準(zhǔn)蛋白質(zhì)的分子量為y,求回歸方程y=f(x),計算顯色條帶的分子量。結(jié)果顯示兔抗血清和豚鼠抗血清均與淋病奈瑟菌裂解物有明顯的反應(yīng)條帶,而非免疫血清則無明顯反應(yīng)。細(xì)菌裂解物經(jīng)過SDS-PAGE分離再經(jīng)考馬斯亮藍(lán)染色后顯示出多條藍(lán)色條帶,其中一條的位置對應(yīng)于Western blot的反應(yīng)條帶,根據(jù)回歸方程求得其分子量為37 300。 (3)抗LD1-KLH多克隆抗體交叉反應(yīng):用ELISA法檢測兔抗血清和豚鼠抗血清與淋病奈瑟菌、金黃色葡萄球菌、甲型溶血性鏈球菌、乙型溶血性鏈球菌、肺炎鏈球菌、大腸桿菌和白色念珠菌等不同微生物之間的交叉反應(yīng)。結(jié)果顯示無論是兔免疫血清還是豚鼠免疫血清均能與淋病奈瑟菌反應(yīng),而與其它參與檢測的微生物之間無交叉反應(yīng)。 二、淋病奈瑟菌原核表達(dá)載體pGEX-PIII的構(gòu)建和鑒定 常規(guī)酚-氯仿法提取淋病奈瑟菌基因組DNA,根據(jù)淋病奈瑟菌外膜蛋白PIII基因序列設(shè)計一對攜帶限制性核酸內(nèi)切酶(BamH I, EcoR I)位點的引物,采用PCR方法獲得PIII全基因片段,產(chǎn)物經(jīng)酚:氯仿:異戊醇抽提后以乙醇沉淀純化;純化產(chǎn)物和載體質(zhì)粒pGEX-3X分別經(jīng)BamH I,EcoR I酶切后行凝膠電泳回收目的片段和質(zhì)粒大片段,經(jīng)T4連接酶連接;連接產(chǎn)物轉(zhuǎn)化到感受態(tài)細(xì)胞中,經(jīng)氨芐青霉素抗性、雙酶切和測序篩選出陽性克隆,命名為pGEX-PIII。 結(jié)論:將淋病奈瑟菌外膜蛋白PIB抗原表位和外膜蛋白PIII聯(lián)合應(yīng)用于淋病檢測試劑的研究在國內(nèi)未見報導(dǎo)。本研究利用人工合成多肽制備針對淋病奈瑟菌特異性多克隆抗體,并用ELISA和Western blot檢測其特異性。結(jié)果表明人工合成多肽具有免疫原性,能誘導(dǎo)機(jī)體產(chǎn)生識別淋病奈瑟菌抗原表位的特異性抗體。PIII的氨基酸殘基數(shù)是PIB抗原表位的數(shù)十倍,用人工合成的方法來獲得將大大增加研究或生產(chǎn)成本。因此,本研究用分子生物學(xué)技術(shù)成功構(gòu)建淋病奈瑟菌外膜蛋白PIII原核表達(dá)載體pGEX-PIII,為進(jìn)一步獲得免疫血清作準(zhǔn)備�？傊�,本研究的工作為新型淋病奈瑟菌快速診斷試劑的研制提供實驗數(shù)據(jù)。
[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 擄 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
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2007
【分類號】：R759.2;R392

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