發(fā)布時(shí)間：2018-05-31 07:23

  本文選題：人乳頭瘤病毒 + 假病毒　； 參考：《廈門大學(xué)》2008年碩士論文

【摘要】： 人乳頭瘤病毒(HPV)能夠引起包括宮頸癌在內(nèi)的多種生殖道疾病,嚴(yán)重危害人類健康。HPV具有嚴(yán)格的宿主特異性和組織分化依賴性,難以通過常規(guī)組織培養(yǎng)方法擴(kuò)增,也難以從病變組織中分離獲得足夠的病毒。因此建立簡(jiǎn)便、高效的HPV體外感染模型是發(fā)展HPV預(yù)防疫苗與治療藥物的迫切需要。近來研究發(fā)現(xiàn)HPV假病毒可用于在體外有效模擬HPV的感染,以此基礎(chǔ)建立的中和實(shí)驗(yàn)方法具有較突出準(zhǔn)確、便捷的優(yōu)點(diǎn),在HPV預(yù)防疫苗、中和抗體研究中顯示出巨大的應(yīng)用潛力。HPV假病毒中和實(shí)驗(yàn)方法的廣泛使用需要進(jìn)一步提高假病毒構(gòu)建效率,同時(shí)能夠滿足高通量檢測(cè)的需要。本實(shí)驗(yàn)室已應(yīng)用磷酸鈣多質(zhì)粒共轉(zhuǎn)染方法在293FT細(xì)胞中建立了構(gòu)建HPV假病毒的方法,本研究以此為基礎(chǔ),進(jìn)一步對(duì)HPV假病毒構(gòu)建體系進(jìn)行系統(tǒng)改進(jìn)和優(yōu)化,建立了更為高效且適合高通量檢測(cè)的HPV中和實(shí)驗(yàn)方法。 建立簡(jiǎn)便高效、高通量的假病毒中和實(shí)驗(yàn)檢測(cè)方法對(duì)提高實(shí)驗(yàn)效率,滿足大規(guī)�？贵w檢測(cè)的需要具有十分重要的意義。同時(shí),建立多個(gè)型別的HPV假病毒中和實(shí)驗(yàn)?zāi)Ｐ蜑榫哂懈弑Ｗo(hù)力的多價(jià)疫苗的開發(fā)提供了全面的中和抗體評(píng)價(jià)體系。 本研究首先探討了在HPV假病毒構(gòu)建體系中優(yōu)化質(zhì)粒轉(zhuǎn)染比例以及磷酸鈣轉(zhuǎn)染條件對(duì)假病毒構(gòu)建效率的影響。結(jié)果顯示,在該體系中不同的HPV結(jié)構(gòu)蛋白表達(dá)質(zhì)粒轉(zhuǎn)染比例對(duì)于假病毒構(gòu)建效率有顯著影響,HPV主要結(jié)構(gòu)蛋白L1的表達(dá)水平是影響構(gòu)建效率的主要因素,L2表達(dá)質(zhì)粒的用量過高或過低均不利于假病毒的構(gòu)建。報(bào)告質(zhì)粒的用量對(duì)假病毒構(gòu)建效率的影響相對(duì)較小。綜合比較顯示,在該體系中L1和L2表達(dá)質(zhì)粒和報(bào)告質(zhì)粒以合適比例(1:1/10:1/2)進(jìn)行共轉(zhuǎn)染可獲得較理想的構(gòu)建效率。同時(shí)通過優(yōu)化磷酸鈣轉(zhuǎn)染試劑的用量和DNA在磷酸鈣溶液中濃度獲得了較優(yōu)的轉(zhuǎn)染條件。通過優(yōu)化改進(jìn),本研究建立了更為高效的HPV假病毒大量制備方法,在HPV16、HPV18、HPV6、HPV11假病毒構(gòu)建中的應(yīng)用結(jié)果顯示相比原方法可顯著提高構(gòu)建效率10～20倍。為HPV假病毒中和實(shí)驗(yàn)的規(guī)�；瘧�(yīng)用提供了有利條件。 為適應(yīng)高通量檢測(cè)的需要,本研究通過改進(jìn)報(bào)告基因及檢測(cè)方法建立了新型的HPV假病毒中和實(shí)驗(yàn)體系。本研究選擇半乳糖苷酶作為報(bào)告基因,構(gòu)建了攜帶優(yōu)化后的半乳糖苷酶表達(dá)元件的HPV假病毒,研究結(jié)果顯示,假病毒感染后的細(xì)胞加入顯色底物后可呈現(xiàn)顯著的藍(lán)色,并且該標(biāo)記可為酶聯(lián)免疫圖像分析系統(tǒng)(Elispot)所檢測(cè)。酶聯(lián)免疫圖像分析系統(tǒng)可對(duì)顯色后的96孔細(xì)胞培養(yǎng)板進(jìn)行連續(xù)快速的自動(dòng)掃描并計(jì)數(shù),滿足了高通量檢測(cè)的需要。本研究進(jìn)一步對(duì)顯色方法進(jìn)行優(yōu)化,提高了檢測(cè)效率。通過綜合應(yīng)用優(yōu)化的半乳糖苷酶報(bào)告基因表達(dá)元件與酶聯(lián)免疫圖像分析系統(tǒng),本研究建立新型的可滿足高通量檢測(cè)要求的HPV假病毒中和實(shí)驗(yàn)方法。本研究應(yīng)用該方法對(duì)四型HPV中和單抗的鑒定結(jié)果表明,半乳糖苷酶假病毒中和實(shí)驗(yàn)方法與目前應(yīng)用的EGFP假病毒中和實(shí)驗(yàn)方法的檢測(cè)靈敏度相當(dāng),同時(shí)新方法還具有可自動(dòng)連續(xù)檢測(cè)、操作簡(jiǎn)便、結(jié)果顯示直觀的優(yōu)點(diǎn),可更好地滿足大規(guī)模中和抗體檢測(cè)實(shí)驗(yàn)的需要。本研究應(yīng)用該方法鑒定獲得了2株HPV16中和單抗,3株HPV18中和單抗,以及HPV6和HPV11中和單抗各5株;同時(shí)對(duì)本中心生產(chǎn)的HPV16、HPV18、HPV6和HPV11四價(jià)VLP疫苗的抗體保護(hù)性進(jìn)行了評(píng)價(jià),為臨床實(shí)驗(yàn)階段所需疫苗的合適劑量提供了參考。 HPV58和HPV52是中國(guó)較為流行的高危HPV型別,但一直以來研究較少。建立HPV58和HPV52假病毒中和實(shí)驗(yàn)?zāi)Ｐ蛯?duì)于研究HPV58和HPV52以及預(yù)防疫苗的開發(fā)具有重要意義。我們將HPV58和HPV52的衣殼蛋白表達(dá)序列進(jìn)行密碼子人源化優(yōu)化后,克隆到表達(dá)載體上,與報(bào)告質(zhì)粒分別共轉(zhuǎn)染293FT獲得了感染性的HPV58和HPV52假病毒。將質(zhì)粒通過尾靜脈高壓注射方式免疫小鼠,成功在小鼠體內(nèi)誘導(dǎo)了高滴度的HPV58和HPV52的中和性抗體。Western blotting結(jié)果顯示這兩型假病毒在約55KD處均具有L1蛋白活性。多抗血清交叉中和實(shí)驗(yàn)表明HPV58和HPV52、HPV16和HPV58之間存在一定水平的抗體交叉保護(hù),而HPV16和HPV52之間沒有可檢測(cè)的抗體交叉保護(hù)性。
[Abstract]:Human papillomavirus (HPV) can cause a variety of reproductive tract diseases, including cervical cancer, which seriously endangers human health and.HPV has strict host specificity and tissue differentiation dependence. It is difficult to expand by conventional tissue culture method, and it is difficult to isolate enough virus from the pathological tissue. Therefore, a simple and efficient HPV in vitro is established. The infection model is an urgent need for the development of HPV vaccine and therapeutic drugs. Recent studies have found that HPV pseudo virus can be used to effectively simulate HPV infection in vitro. The neutralization experiment based on this method has the advantages of prominent accuracy and convenience. It has shown great potential application potential of.HPV false disease in HPV vaccine and anti body research. The extensive use of toxic and experimental methods needs to further improve the efficiency of pseudo virus construction and meet the needs of high throughput detection. This laboratory has established a method of constructing HPV pseudo virus in 293FT cells by using calcium phosphate multi plasmids co transfection method. This study is based on this and further studies the construction of HPV pseudo virus. System improvement and optimization, a more efficient and high-throughput HPV neutralization test method has been established.
The establishment of a simple, efficient, high throughput test method for pseudo virus neutralization test is of great significance to improve the efficiency of the experiment and meet the needs of large-scale antibody detection. At the same time, the establishment of HPV pseudo virus neutralization experiment model of multiple types provides a comprehensive neutralization antibody evaluation system for the development of high protective polyvalent vaccines.
In this study, the effect of optimized plasmid transfection ratio and calcium phosphate transfection conditions on the efficiency of pseudo virus construction in the HPV pseudo virus construction system was investigated. The results showed that the transfection ratio of different HPV structure protein plasmids in the system had significant influence on the efficiency of pseudo virus construction, and the expression level of the main structural protein L1 of HPV was The main factors affecting the construction efficiency, the high or low dosage of the L2 expression plasmid are not conducive to the construction of the pseudo virus. The effect of the dosage of the reported plasmid on the construction efficiency of the pseudo virus is relatively small. The comprehensive comparison shows that the L1 and L2 expression plasmids and the reported plasmids can be co transfected with a suitable ratio (1:1/10:1/2) in the system. At the same time, better transfection conditions were obtained by optimizing the dosage of calcium phosphate transfection reagent and the concentration of DNA in calcium phosphate solution. Through optimization and improvement, a more efficient method for preparing HPV pseudo virus was established. The application results in the construction of HPV16, HPV18, HPV6, HPV11 pseudo virus showed that the original method was significant compared with the original method. The construction efficiency is increased 10~20 times, which provides favorable conditions for the large-scale application of HPV virus neutralization test.
In order to meet the needs of high throughput detection, a new type of HPV pseudo virus neutralization experiment system was established by improving the reporter gene and detection methods. This study selected galactosidase as a reporter gene and constructed a HPV pseudo virus carrying the optimized galactosidase expression element. The results showed that the cells after the false virus infection were added to the virus. The chromogenic substrate can present a significant blue color, and the marker can be detected by the enzyme linked immunoimage analysis system (Elispot). The enzyme linked immunoimage analysis system can continuously and quickly automatically scan and count the 96 pore cell culture plates after the color display, and meet the needs of high throughput test. This study further studies the color rendering method. In this study, a new HPV pseudo virus neutralization experiment was established to meet the requirements of high throughput detection. This method was applied to the identification of four type HPV neutralization McAbs and the results of this study showed that galactoside was used for the identification of galactoside. The enzyme pseudo virus neutralization test method is similar to the detection sensitivity of the EGFP pseudo virus neutralization test method, and the new method also has the advantages of automatic continuous detection, simple operation and direct display, which can better meet the needs of large-scale neutralization antibody test. 2 HPV1 strains were identified by this method. 6 neutralization mAb, 3 HPV18 neutralization McAbs, and 5 strains of HPV6 and HPV 11 and McAb respectively. The antibody protection of HPV16, HPV18, HPV6 and HPV11 tetravalent VLP vaccines produced in this center was evaluated to provide reference for the appropriate dosage of the vaccine needed in the clinical trial stage.
HPV58 and HPV52 are the most prevalent high risk HPV types in China, but have been less studied. The establishment of HPV58 and HPV52 pseudo virus neutralization experimental models is of great significance for the study of HPV58 and HPV52 and the development of vaccine prevention. We have optimized the expression sequence of the capsid protein of HPV58 and HPV52 and cloned the expression load. In vivo, the infected HPV58 and HPV52 pseudo virus were obtained by CO transfection of 293FT with the reported plasmid. The plasmid was immunized with the tail vein high pressure injection in mice. The high titer HPV58 and HPV52 neutralizing antibody.Western blotting results were successfully induced in mice. The results showed that the two types of false viruses had L1 protein activity at about 55KD. The cross neutralization test of polyclonal sera showed that there was a certain level of cross protection between HPV58 and HPV52, HPV16 and HPV58, and there was no detectable cross protection between HPV16 and HPV52.
【學(xué)位授予單位】：廈門大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R373;R737.33

【共引文獻(xiàn)】

相關(guān)期刊論文 前1條

1 林曉華;吳寧;侯麗輝;吳效科;;人乳頭瘤病毒與宮頸腫瘤關(guān)系的研究進(jìn)展[J];醫(yī)學(xué)研究生學(xué)報(bào);2006年02期

相關(guān)博士學(xué)位論文 前2條

1 范江濤;HPV16疫苗的聯(lián)合免疫研究[D];廣西醫(yī)科大學(xué);2006年

2 趙欣;人白細(xì)胞介素15 cDNA協(xié)同優(yōu)化的人乳頭瘤病毒16型E7核酸疫苗治療宮頸癌的初步研究[D];中國(guó)協(xié)和醫(yī)科大學(xué);2007年

相關(guān)碩士學(xué)位論文 前3條

1 許恬怡;泌乳素調(diào)節(jié)的NK-92細(xì)胞抗人子宮頸癌細(xì)胞體內(nèi)外研究[D];安徽醫(yī)科大學(xué);2006年

2 姜育q，

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