A Method For The Amplification Of Full-length Envelope Genes Of HIV-1among MSM Population In Beijing

ObjectiveDue to the high variability of HIV, there is still no effective vaccine found atpresent. More research of the virus structure and immune response is needed forprevention and treatment. The envelope protein is the main target of neutralizingantibodies against HIV. This protein play an important role on the combination andintegration of HIV virus and host cells. The full-length envelope genes contain3000bp, so it is difficult to amplify this gene by a conventional PCR method. This topicaims to explore the method for amplification of the full-length envelope genes fromplasma and blood.Material and Methods1. Sample: The HIV infectors whom are MSM in Beijing will be chosen. Theviral load in plasma of these people should be higher than the detection limit.theirplasma samples should against broad range of neutralizing antibodies.2. The extraction of HIV virus nucleic acid and the amplification of env geneUsing the DNA/RNA extraction kit extract the nucleic acid of HIV. Then, weamplify it by using RevenvA+ENV-lo and RevenvB-TOPO+ENV-lo as the firstand second round PCR primers in DNA samples; In RNA samples,we need SG3-up+SG3-lo+RevenvA and RevenvB-TOPO+ENV–lo as RT-PCR and second roundPCR primers for amplification.3. PCR products purificationThe PCR Products were purified with QIAquick Gel Extraction Kit．4. Add an “A” to the gene and purify the productsMix the Products (from step3,40.5ul) with10×Buffer(5ul), dATPs (4ul) andTEQase(0.5ul), then, let those react at72℃for20minutes.5. Connect the gene to pMD18-T vector Mix the product (from step4,4ul) with pMD18-T vector(1ul) and Solution I(5ul), then, let those react at16℃for whole night.6. Transform the purpose gene into the cellsMix the products (from step5,10ul) with the competent cells, then, place it onthe ice for30minutes. Put it in the Constant temperature water bath pot for hot shot at42℃in90seconds.7. CloneAdd the product (from step6) to LB liquid medium, and clone it in cultureincubator for1hour. Then spread the cultures (200ul) on LB solid medium, clone it at37℃for whole night.8. Cultures identificationPick the colony in the LB tablet which colony diameter is about1-2mm, the addthem into LB liquid medium (Amp,1ml) and clone it. Then, identify the bacteria byPCR.Results1. Sample characteristicsWe have42cases satisfied the conditions, in which,24cases extract DNA,29cases extract RNA,and11cases extract both. The average age of samples are31.02±6.82years old.According to the viral load of samples, we can be divided them intotwo groups, the high viral load group, which have25cases, and the low viral loadgroup, which have17cases.2. The method for amplification of envelope protein gene of HIV virus isestablished.In this research, we have amplified out22cases, positive rate is52.4%. We haveamplified out15cases in DNA samples, positive rate is62.5%,15cases in RNAsamples, positive rate is51.7%.3. The HIV envelope protein gene T-A cloning and sequence analysisWe build a gene evolutionary tree of HIV env gene by Mega5.0. Then, we findthat this batch of sample mainly comes from HIV subtype AE and subtype B/C. We also find that the discrete rate is small between samples of the same subtype genes.Conclusion1. We established a method to amplify the full-length envelope genes fromplasma and blood sample. Our primers cover the main three popular HIV subtypes.2. There is virus no significant differences between the DNA sample and theRNA sample in amplification rate of the full-length envelope genes.3. Samples in different viral load level have different amplification efficiency.Samples with high viral load level have a higher positive rate than the low viral loadlevel ones.4. Specific primers can improve the efficiency of the amplification rate of thefull-length envelope genes.