Development Of Anti-HIV Tradiontional Chinese Medicine And Antibodies Screening System Based On CCR5 Receptor

Background: AIDS,also known as Acquired Immune Deficiency Syndrome,is a result of infection with human immunodeficiency virus type 1(HIV-1)leading to complete loss of immune function of the body of infectious diseases.According to the National Health and Family Planning Commission’s data,.in 2016 there are almost 62 million AIDS patients in China,which has brought great risks for public health and social stability.HIV-1 treatment has been a worldwide problem.At present,the main clinical use of reverse transcription inhibitors,integrase inhibitors,protease inhibitors,but the current treatment can not completely eliminate the virus to cure AIDS.And the current antiviral treatment is susceptible to drug resistance and serious side effects limit its development,so the development of safe and effective HIV-1 vaccine is a new direction for HIV prevention and control.HIV-1 virus has the characteristics of high mutation and gene recombination.HIV vaccine research is facing great difficulties and challenges.CCR5(The C-C chemokine receptor type 5)receptor is one of the indispensable co-receptor for clinically existing HIV-1 strains,making it an important target for antiretroviral therapy.CCR5,as an intrinsic receptor in host cells,plays an important role in HIV infection and is highly conserved,and is well suited as an entry point for anti-HIV-1 studies.In this study,two aspects of CCR5 receptors were mainly studied.On the one hand,the expression of CCR5 protein was detected by flow cytometry by adding traditional Chinese medicine extract to the cell line stably expressing CCR5.According to the expression of CCR5 in vitro to determine the role of traditional Chinese medicine extract on the CCR5 receptor and anti-HIV-1 activity;On the other hand,the mammalian two-hybrid system was used to study the interaction between anti-HIV-1 neutralizing antibody PRO140 and host cell CCR5 gene,which laid the foundation for further screening and study of CCR5 antibody.Objective: To investigate the effect of traditional Chinese medicine extract on the expression of CCR5 protein in vitro and to determine whether the traditional Chinese medicine has potential anti-HIV-1 activity.To construct plasmid pVP16-PRO140 containing the neutralizing antibody gene,the recombinant plasmid and the plasmid containing CCR5 Were co-transfected into CHO cells,analyzed by the reporter gene analysis,whether the two proteins interact,lay the foundation for further antibody research.Methods: 1.Add the traditional Chinese medicine extract to the cell line that stably express CCR5 protein.The expression of CCR5 was detected by flow cytometry by the extract of traditional Chinese medicine.2.The recombinant plasmid pVP16-PRO140 was constructed and inserted into the vector pVP16 by plasmid extraction,digestion and gel recovery.The recombinant plasmid was confirmed by colony PCR,restriction enzyme electrophoresis and sequencing.3.Recombinant plasmid pM-CCR5 was constructed by molecular cloning and transfected into CHO cells by liposome.The recombinant plasmid pM-CCR5 and empty vector pM were transfected into CHO cells,and then the expression of CCR5 gene was detected from the protein level by Western Blot.4.Transfection of plasmid pVP16-PRO140 and plasmid pM-CCR5 and reporter gene into CHO cells through a mammalian two-hybrid system.5.The effect of the interaction between the PRO140 antibody and the CCR5 receptor was identified using secreted alkaline phosphatase assays.Conclusion: 1.In vitro environment,the extract of traditional Chinese medicine andrographolide and quercetin can inhibit the expression of CCR5 protein in different degree,and the expression of CCR5 protein in rutin and Atractylodes macrocephala has no obvious effect on the expression of CCR5 protein.The mammalian two-hybrid system was initially constructed and the interaction between CCR5 protein and single-chain antibody PRO140 was verified.It was proved that the system could be used to detect whether the CCR5 protein interacted with the target protein and target the antibody of CCR5 receptor.