本文關(guān)鍵詞： HIV 包膜蛋白基因全長擴(kuò)增 聚合酶鏈反應(yīng)　出處：《首都醫(yī)科大學(xué)》2013年碩士論文　論文類型：學(xué)位論文

【摘要】：研究目的 由于HIV病毒的高變異性，目前仍無有效的疫苗能夠防治該疾病。為了制定對(duì)該疾病的更好的預(yù)防及治療措施，需對(duì)HIV病毒基因、結(jié)構(gòu)及與機(jī)體相互間的免疫反應(yīng)進(jìn)行進(jìn)一步研究。HIV病毒的結(jié)構(gòu)基因中的包膜蛋白是HIV中和抗體作用的主要靶蛋白，其在HIV病毒與宿主細(xì)胞結(jié)合及融合方面起重要作用。由于該基因全長序列約3000bp，所以用常規(guī)的PCR方法較難得到其全長產(chǎn)物片段。 本課題旨在探尋從血漿及全血樣本中擴(kuò)增HIV病毒env全長基因序列的方法，針對(duì)我國特有的HIV流行亞型，，為下一步深入研究包膜蛋白抗原的嗜性、抗原性、免疫原性及中和敏感性等建立較為成熟的env全長基因擴(kuò)增技術(shù)平臺(tái)。 研究材料與方法 一、研究對(duì)象：選取北京地區(qū)男男同性戀人群中的已確證感染HIV病毒者。同時(shí)這些感染者需滿足病毒載量可測得及血漿具有廣泛中和抗體活性的條件。 二、研究方法 1、HIV病毒核酸提取及病毒env基因擴(kuò)增 取全血樣本白膜層200ul、血漿樣本140ul分別使用Qiagen公司的DNA提取試劑盒及RNA提取試劑盒提取病毒核酸。獲取病毒核酸后，對(duì)DNA樣本，分別對(duì)以RevenvA+ENV-lo及RevenvB-TOPO+ENV-lo為第一、二輪PCR引物進(jìn)行擴(kuò)增；對(duì)RNA樣本分別以SG3-up+SG3-lo+RevenvA及RevenvB-TOPO+ENV-lo為RT-PCR及第二輪PCR引物進(jìn)行擴(kuò)增。 2、PCR產(chǎn)物切膠純化回收 將上述PCR產(chǎn)物使用Qiagen公司PCR產(chǎn)物純化回收試劑盒進(jìn)行純化回收。 3、目的基因片段平末端加A并純化回收 取上述純化回收產(chǎn)物40.5ul，與5ul的10×Buffer、4ul的dATPs及0.5ul的TEQase配成50ul體系，于72℃反應(yīng)20分鐘。 4、與pMD18-T vector連接 取上述產(chǎn)物4ul，與1ul的pMD18-T vector及5ul的Solution I配成10ul體系，于16℃過夜。 5、目的基因轉(zhuǎn)化入感受態(tài)細(xì)胞 取10ul上述產(chǎn)物加入感受態(tài)細(xì)胞懸液中，冰上放置30分鐘；恒溫水浴鍋中42℃熱擊90秒。 6、單克隆培養(yǎng) 將上述產(chǎn)物加入LB液體培養(yǎng)基中，恒溫箱中培養(yǎng)1小時(shí)。后取培養(yǎng)液200ul涂于LB固體培養(yǎng)基上，于37℃過夜培養(yǎng)。 7、單克隆質(zhì)粒鑒定 挑取上述LB平板中直徑約1-2mm的單個(gè)菌落，于1ml LB液體培養(yǎng)基中（含Amp）中過夜培養(yǎng)，后取菌液進(jìn)行PCR鑒定。 研究結(jié)果 1、樣本基本特征 入組樣本42例，提取DNA、RNA分別24例及29例，均提取有11例。樣本平均年齡為31.02±6.82歲。根據(jù)病毒載量高低將樣本分為兩組，高病毒載量組25例；低病毒載量組17例。 2.HIV病毒包膜蛋白全長基因擴(kuò)增方法的建立 在本實(shí)驗(yàn)中，擴(kuò)增出病毒包膜蛋白基因22例，陽性率為52.4%。其中DNA標(biāo)本中擴(kuò)增出15例，陽性率為62.5%；RNA標(biāo)本中擴(kuò)增出15例，陽性率為51.7%。 3.HIV病毒包膜蛋白基因T-A克隆及序列分析 用Mega5.0構(gòu)建部分樣本HIV病毒env全長基因進(jìn)化樹，發(fā)現(xiàn)此批樣本主要來源于AE亞型和B/C亞型，且同一亞型的樣品之間基因離散率較小。 結(jié)論 1、建立了從血漿、全血細(xì)胞樣本來源的病毒RNA及DNA擴(kuò)增env全長基因的方法，引物涵蓋我國主要三個(gè)HIV流行亞型。 2、HIV病毒DNA及RNA樣本在病毒env全長基因擴(kuò)增陽性率沒有顯著差異。 3、病毒env基因的擴(kuò)增效率受病毒載量影響，高病毒載量的樣本陽性率高。 4、亞型特異性引物的使用能提高相應(yīng)病毒亞型的env基因擴(kuò)增效率。
[Abstract]:research objective
Due to the high variability of the HIV virus, there is still no effective vaccine can prevent and cure the disease. In order to formulate the measures of prevention and treatment of the disease better, gene of HIV virus, and the structure and immune response between structural gene research of.HIV virus in the envelope protein is the major target protein HIV effect of neutralizing antibodies, plays an important role in HIV binding and fusion of virus and host cells. The gene sequence is about 3000bp, so the conventional PCR method is difficult to get the full-length fragment.
The purpose of this study is to explore the method of amplification of full-length sequence of HIV virus Env from plasma and whole blood samples, according to China's unique HIV subtypes, for the further research of envelope protein antigen tropism, antigenicity, immunogenicity and neutralization sensitivity of env gene to establish mature amplification technology platform.
Research materials and methods
Object of study: select the confirmed HIV infected HIV virus in Beijing gay men. At the same time, these infected persons need to meet the requirements of viral load and the neutralizing antibody activity of plasma.
Two, research methods
1, HIV virus nucleic acid extraction and virus env gene amplification
Whole blood samples albuginea 200ul, plasma samples were used 140ul Qiagen DNA extraction kit and RNA extraction kit. After obtaining the viral nucleic acid virus nucleic acid of DNA samples, respectively, on RevenvA+ENV-lo and RevenvB-TOPO+ENV-lo for the first, second round of PCR primers were amplified; of RNA samples respectively by SG3-up+SG3-lo+RevenvA and RevenvB-TOPO+ENV-lo RT-PCR and the two round PCR primers were amplified.
2, PCR product gels purification recovery
The PCR products were purified and recovered by using the PCR product purification recovery kit of Qiagen company.
3, the end of target gene fragment with A and purification and purification
The purified recovery product 40.5ul was used as 50ul system with 5ul's 10 x Buffer, 4ul dATPs and 0.5ul TEQase, and reacted at 72 centigrade for 20 minutes.
4, connect with pMD18-T vector
The above product, 4ul, was matched with the pMD18-T vector of 1ul and Solution I of 5ul to be a 10ul system at 16 C for the night.
5, the target gene is transformed into the receptive cell
The 10ul products were added to the sensory cell suspension and placed on the ice for 30 minutes. At the constant temperature water bath, the heat shock was 42 centigrade for 90 seconds.
6, McAb culture
The above products were added to the LB liquid medium for 1 hours in the incubator. Then the culture medium was applied to the solid medium of LB and cultured at 37 C for the night.
7, identification of monoclonal plasmids
Single colonies of the LB tablet in the diameter of 1-2mm, 1ml in LB liquid medium (containing Amp) in cultured overnight, after bacteria identified by PCR.
Research results
1, the basic feature of the sample
42 cases were enrolled in the study. DNA and RNA were extracted from 24 cases and 29 cases respectively. 11 cases were extracted. The mean age of the samples was 31.02 + 6.82 years. According to the viral load, the samples were divided into two groups, 25 cases of high viral load group, and 17 cases of low viral load group.
The establishment of full length gene amplification method for 2.HIV virus envelope protein
In the experiment, 22 cases of viral envelope protein gene were amplified, the positive rate was 52.4%., among which 15 cases were amplified in DNA samples, the positive rate was 62.5%, and 15 cases were amplified in RNA samples, the positive rate was 51.7%..
Cloning and sequence analysis of 3.HIV virus envelope protein gene T-A
Mega5.0 was used to construct partial sample HIV virus Env full-length gene evolution tree. It was found that this batch of samples mainly came from AE subtype and B/C subtype, and the gene dispersion rate between the same subtype was smaller.
conclusion
1, a method of amplification of the full-length env gene from the virus RNA and DNA from the plasma and whole blood cell samples was established. The primers covered the three major HIV subtypes of our country.
2, there was no significant difference in the positive rate of the full length gene amplification of the DNA and RNA samples of the HIV virus in the virus Env.
3, the amplification efficiency of the virus env gene is affected by the viral load, and the positive rate of the high viral load is high.
4, the use of subtype specific primers can improve the amplification efficiency of the env gene of the corresponding virus subtype.

【學(xué)位授予單位】：首都醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R512.91

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