Study On Construction, Expression, Activity Of New Anti-HIV-1 Protein Cyanovirin-N, Griffithsin And Recombinant Directing Agents

Human immunodeficiency virus type 1 (HIV-1) is the aetiological agent of acquired immune deficiency syndrome (AIDS). Since the first case infected with HIV was reported in Los Angeles in 1981, the research of HIV-1 has never been paused. Howere, until now, there are still no effective vaccines or drugs according to the prophylaxis or therapy of this disease. Although the widespread use of regimen, highly active antiretroviral therapy (HAART), have maximally reduced the levels of circulating virus, significantly decreased the rates of the opportunistic infections and dramatically improved the survival and quality of life of HIV-infected individuals, HIV cannot yet be eradicated from infected individuals. And, HAART have some unexpected side- effects such as high cost of long-term administration, the evolution of drug-resistant variants, the virus rebound in viremia after withdraw medication, etc., all the above limit its use. So, new therapy methods as well as agents are required.As a new research field, directing therapy has achieved gratifying results in cancer therapy. Given the similar between AIDS and cancer, the methods has broad application prospect on AIDS treatment. The previously research shows that directing agent targeted to HIV-1 envelope protein could kill the infected cells specifically. Therefore, it can be used as a good adjuvant agent of HAART. However, there are still some problems need to be considered among AIDS directing therapy such as the target tools, biological materials, high mutation rate of HIV envelope protein, the reconstruction of immune system, etc.In view of the current problems that mentioned above on AIDS/HIV treatment, this paper focus on the constructing an agents that could kill HIV infected cells specifically as well as have immune therapy function. Cyanovirin N (CVN) and Griffithsin (GRFT) were selected. They can bind to HIV envelope specically. Additionally, Staphylococcus aureus extoxin A (SEA) which has immune inducing activity was selected.The sequence of CVN, GRFT and SEA were obtained from GenBank. Their codons were modified and optimized for optimal expression in E.coli and/or yeast, then were synthesized by chemical method respectively. Both CVN and GRFT were cloned into protokaryon vector pET-28 a(+). Recombinant expressioin plasmid pET-C and pET-G were constructed. Through IPTG induction, the proteins were expressed well by SDS-PAGE analysis and Western Blot identification.Then, SEA gene was amplified by PCR, and sequence of Linker (Gly4Ser)3 was added downstream of SEA. Fusion genes of recombinant directing agents CS and GS were constructed by inserting CVN or GRFT gene into the downstream of SEA respectively. Both of them were cloned into protokaryon vector pET-28 a(+). Recombinant expressioin plasmid pET-S, pET-CS and pET-GS were constructed. After IPTG induction, SDS-PAGE and western blot analysis showed that the proteins were expressed successfully.After optimizing the induction conditions including inducing temperature, inducing time and IPTG concentration, the best expression conditions of five proteins were decided. CVN and GRFT shared the same conditions, namely, they were induced 4 hours at 37℃while IPTG concentration is 0.8 nM. But the others have different induction conditions.Under the optimal induction condition, the highest products of CVN, GRFT, SEA, CS, GS account for 46.4%, 55.84%, 25.15%, 38.83%, and 32.3% of the total bacterial proteins respectively. They can reached approximatelly a yield of 0.93mg/mL, 1.31mg/mL, 0.19mg/mL, 0.37mg/mL, and 0.29mg/mL respectively. And all the different garget products all existed in form of inclusion body.Products in form of inclusion body were eluted by different concentration of urea. Unexpected proteins could be removed partly. Then the eluted products were renatured and purified by gradient gel filtration chromatography. The purity of the protein could reach 98.8% and 10.39mg protein could be obtained from 100mL bacteria media after renaturing. By Dot-ELISA analysis, the proteins have good binding activities. The results indicated that the proteins were renatured successfully.To investigate the cell binding activity and CTLs mediated by recombinant directing agents, the recombinant plasmid pD-160,pD-120,pD-41 were constructed on the base of eukaryotic expression vector pDisplay. After transfected into HeLa cells and screened by G418, identificated by SDS-PAGE, western blot and indirect immuno fluorescence assay (IFA) of HIV-1 gp160 and gp120 cell, the stable cell models expressing proteins were obtained. The cell model can mimic HIV-1 infection.By using the cell models, we detected the cell binding activities of four proteins by ELISA. The specific killing activity of CTLs mediated by recombinant directing agents was detected with Cytotox 96 Non-Radioactive Cytotoxicity Assay, taking CVN, GRFT as control. The results showed the recombinant directing agents had good cell binding activities and could nonspecifically activate PBMCs and specifically mediate CTLs to kill the target cells expressing HIV-1 gp120 proteins, which can be used as a good adjuvant agent for HAART therapy.Finally, in order to obtain biological activity protein directly, Pichia pastoris, a eukaryotic expression system was used too. Four yeast expression plasmids of pPIC9K-C, pPIC9K-G, pPIC9K-CS and pPIC9K-GS were constructed using yeast secretory expression vector pPIC9K. After linearized the recombinant plasmids, they were transformed into the competent yeast strain GS115 of Pichia pastoris by electronic method respectively. The positive transformants were selected and identified by MD/MM plate, PCR, G418 screening. After that, they were induced by methanol. SDS-PAGE analysis, western blot identification and Dot-ELISA test revealed that the proteins were expressed and had good biding activities. Thess experiments laid a good foundation for the future scale production of proteins.The study will be hopelly exceed the bottlenecks of AIDS/HIV treatment. It will provide the original information and technical data for development of a new HAART adjuvant therapy agent. Furthmore, the research will provide good research ideas and evidence for other chronic diseases (such as HBV) as well as enveloped virus (such as the Ebola virus, SARS virus, Bird flu Virus, etc.).The study has not been reported before in the country. And it is still at the initial stage in the abroad. Also, some research is the first time reported at the international level.