Construction And Identification Of HIV-1Outer Membrane Protein Cell Model

AIDS,acquired immune deficiency syndrome,the current threat to human life, health and global public health security of the biggest problems, also known as"super cancer" and "century killer".Human immunodeficiency virus (HIV) is the main pathogens.taxonomy it belongs to the retrovirus Branch,lentivirus, the primate lentivirus group.HIV is a virus attacks the human immune system. it the most important T4lymphocytes in the body’s immune system as an attack goal,the destruction of a large number of T4lymphocytes,resulting in the lymphocytes of a large number of failure.lifelong infection of the virus within the Territory,to destroy the human immune balance,so that the human body to become the carrier of various diseases of HIV itself does not cause any disease, but When the immune system is destroyed by HIV,the human resistance is too low, resulting in a variety of opportunistic infections or tumor caused death.The most world outstanding medical researchers have paid great efforts, but has not yet been developed to cure AIDS,effects of drugs,has not developed an effective vaccine for the prevention.At present,the disease mortality is almost as high as100percent,our country has been included in the CPI (B) of Notifiable Diseases,and as one of the frontier health surveillance of infectious diseases. While restricting the anti-HIV effects of drugs and therapeutic vaccines for further development of one of the reasons is the lack of simple and effective test model to evaluate its effect.Cells as a model to detect and avoid the shortcomings of the dangerous and difficult to operate the direct use of strain or organization exists,it is an indispensable link in the currently used to conduct clinical trials.This study,lentiviral shuttle plasmid pLVX,insert outer membrane protein gene of the gp120HIV-1,empty vector as a positive control,combined with lentiviral packaging plasmid pLPl (gag/pol),pLP2(Rev),pLP-VSVG,a total of transfected293T cells,packaged with a view to a single round of infection activity of pseudotyped recombinant lentiviral infected mouse breast cancer cells,the exogenous gene through lentiviral integration into the target cell genome.By G418pressure screening,positive clones cells observed by fluorescence microscopy,PCR, RT-PCR,Western Blot,ELISA to detect the integration of the gp120gene, transcription,expression and binding activity.The results show that successfully build the expression of HIV-1outer membrane protein of lentiviral recombinant plasmid pLV-120, RT-PCR, Western blot and laser scanning confocal microscope, a good expression of the target protein on the cell surface. Successfully packaged with a single round of infection activity pseudotyped recombinant lentivirus LV-120and LV-ZsGreenl complete appearance of virus particles, neat and compact spike, higher virus titers. Infection4T1by PCR and RT-PCR successfully amplified the target band of1596bp, indicating that the gp120gene successful integration of transcription. Bright green fluorescence observed under fluorescent microscope and Western blot to detect the target band of120kDa that GP120protein is a good expression. ELISA results showed that the cell model and the binding activity of recombinant oriented preparations. Through this study, the use of lentiviral vector system successfully able to accurately express the target cells of HIV-1outer membrane protein in vitro Next the development of anti-AIDS/HIV therapeutic vaccines and genetic engineering targeted agents or antivirals cell model, animal model laid the foundation.