北京市MSM人群HIV-1流行亚型包膜蛋白基因全长扩增方法研究

发布时间：2018-02-27 04:34

本文关键词： HIV 包膜蛋白基因全长扩增聚合酶链反应　出处：《首都医科大学》2013年硕士论文　论文类型：学位论文

【摘要】：研究目的由于HIV病毒的高变异性，目前仍无有效的疫苗能够防治该疾病。为了制定对该疾病的更好的预防及治疗措施，需对HIV病毒基因、结构及与机体相互间的免疫反应进行进一步研究。HIV病毒的结构基因中的包膜蛋白是HIV中和抗体作用的主要靶蛋白，其在HIV病毒与宿主细胞结合及融合方面起重要作用。由于该基因全长序列约3000bp，所以用常规的PCR方法较难得到其全长产物片段。本课题旨在探寻从血浆及全血样本中扩增HIV病毒env全长基因序列的方法，针对我国特有的HIV流行亚型，，为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较为成熟的env全长基因扩增技术平台。研究材料与方法一、研究对象：选取北京地区男男同性恋人群中的已确证感染HIV病毒者。同时这些感染者需满足病毒载量可测得及血浆具有广泛中和抗体活性的条件。二、研究方法 1、HIV病毒核酸提取及病毒env基因扩增取全血样本白膜层200ul、血浆样本140ul分别使用Qiagen公司的DNA提取试剂盒及RNA提取试剂盒提取病毒核酸。获取病毒核酸后，对DNA样本，分别对以RevenvA+ENV-lo及RevenvB-TOPO+ENV-lo为第一、二轮PCR引物进行扩增；对RNA样本分别以SG3-up+SG3-lo+RevenvA及RevenvB-TOPO+ENV-lo为RT-PCR及第二轮PCR引物进行扩增。 2、PCR产物切胶纯化回收将上述PCR产物使用Qiagen公司PCR产物纯化回收试剂盒进行纯化回收。 3、目的基因片段平末端加A并纯化回收取上述纯化回收产物40.5ul，与5ul的10×Buffer、4ul的dATPs及0.5ul的TEQase配成50ul体系，于72℃反应20分钟。 4、与pMD18-T vector连接取上述产物4ul，与1ul的pMD18-T vector及5ul的Solution I配成10ul体系，于16℃过夜。 5、目的基因转化入感受态细胞取10ul上述产物加入感受态细胞悬液中，冰上放置30分钟；恒温水浴锅中42℃热击90秒。 6、单克隆培养将上述产物加入LB液体培养基中，恒温箱中培养1小时。后取培养液200ul涂于LB固体培养基上，于37℃过夜培养。 7、单克隆质粒鉴定挑取上述LB平板中直径约1-2mm的单个菌落，于1ml LB液体培养基中（含Amp）中过夜培养，后取菌液进行PCR鉴定。研究结果 1、样本基本特征入组样本42例，提取DNA、RNA分别24例及29例，均提取有11例。样本平均年龄为31.02±6.82岁。根据病毒载量高低将样本分为两组，高病毒载量组25例；低病毒载量组17例。 2.HIV病毒包膜蛋白全长基因扩增方法的建立在本实验中，扩增出病毒包膜蛋白基因22例，阳性率为52.4%。其中DNA标本中扩增出15例，阳性率为62.5%；RNA标本中扩增出15例，阳性率为51.7%。 3.HIV病毒包膜蛋白基因T-A克隆及序列分析用Mega5.0构建部分样本HIV病毒env全长基因进化树，发现此批样本主要来源于AE亚型和B/C亚型，且同一亚型的样品之间基因离散率较小。结论 1、建立了从血浆、全血细胞样本来源的病毒RNA及DNA扩增env全长基因的方法，引物涵盖我国主要三个HIV流行亚型。 2、HIV病毒DNA及RNA样本在病毒env全长基因扩增阳性率没有显著差异。 3、病毒env基因的扩增效率受病毒载量影响，高病毒载量的样本阳性率高。 4、亚型特异性引物的使用能提高相应病毒亚型的env基因扩增效率。
[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.

【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R512.91

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