1.Expression And Identification Of Immuniological Activities A Novel HIV-1 Gp120 And Human Interferon Gamma Fusion Protein 2.Psychologic Problems Of People With HIV-1 And Their Psychosocial Environment And Response In Zhejiang Province, China

AIDS is a kind of serious contagious disease which could destroy the human immune function and lead to death in the last after infecting people. Nowsdays the medicine fields are trying to seek the week points of the virus in itself's defence system so as to control or kill the virus. One of the important targets is the envelop protein of HIV— gpl20.The length of genome is about 9.8kb, and it consists of 9 open reading frames encoding several kinds of viral protein including 3 structure genes(gag,pol,env),2 adjustment genes(tat,rev) and 4 auxiliary genes(vif,vpr,vpu,nef). gp120 is the envelop protein and is encoded by env gene. In the process of viral replication the env gene is firstly expressed into the fore-peptide with the molecule quantity of 160KD, then is broken into two carbohydrate proteins or gp120 and gp41. gp120 has 5 variable regions(V1~V5) and 5 constant regions(C1~C5). It has been known that CD4 structural region in gp120 is a complicated overlap structure which consists of several contact points to CD4 molecule. Among them possibly include two anti-water structure points of C2 and C4 of gp120 and the water structures of C3 and C4. Lots of researches show that four regions of gp120 such as V3 region ,CD4 combination region, V1 region,V2 region are related to neutralization.gp120 contains many antigentic determinants which have strong immunogenicity. The analysis of match among gene sequences shows that the mutation extents of gp120 in different strains are very high. It has been confirmed that the mutation points of gp120 mainly locate in V3 region, and the rest regions are comparatively very constant. In thefields of expression in vitro, the whole length of gpl20 with 1.5kbp can not be expressed steadily and effectively, whereas the protein of N parts with more than 500 bp of gpl 20 ( one third of gpl20 from the beginning of N part, including VI and V2 regions) is expressed steadily and effectively and do not decompose. By confirmation of indirect ELISA and Western blot, the expression products have good antigenicity and specificality, and this gene sequence is comparatively conservative. Considering the widely used cytokine adjuvant in the immune research and therapy on hepatitis B, tumour and other diseases—mainly constituting DNA recombination of antigen and cytokine to strength and change the immune reaction. Therefore, the programme team selects interferon gamma and gpl20 N parts as target and make them constitute recombination. Then, observe wether the recombinant strengths the immune reaction against gpl20 antigen or not. If the experiment result shows it is positive or strength the impact of CTL, it will no doubtly provide worthy research base for clinic treatment on AIDS. Especially the idea is to be based on the impact of cytokine net and then to optimize the specific immune reaction and induce the host to produce the effective immune protection, therefore it is very important to HIV infectants who are with low immune ability and immunodeficiency. ObjectiveTo construct the prokaryotic plasmid that encoding gpl20 N and hlFN- y recombinant gene, express the confusion protein in DE3 cells. Then use the expression protein as antigen to stimulate BALB/c mouse for testing the immune reaction differences especially the cell immune reaction differences between the confusion protein and pure gpl 20 protein. MethodThe N parts of gpl20 gene sequence was got through sub-clone. The prokaryotic expression plasmid pet44b—gpl20 N was constructed by inserting gpl20 N into pet44b.Then the recombinant plasmid was transmitted into DE3 cell in vitro., next it was induced to express in the low temperature. The expressed protein was collected and pured.The eukaryotic expression plasmid pet44b—gpl20 N/hlFN- Y was constructed by inserting the gpl20N/hIFN- Y gene into pet44b by two sub-clone. Then the recombinant plasmid was transmitted into DE3 cell in vitro., next it was induced to express in the low temperature. The expressed protein was collected and pured. Randomly selected and set 3 groups with 20 mouses per group from female and weight equal BALB/c mouse. One group was as control group in which each mouse was injected into PBS with the way of i.s, another one was gpl20 group in which each mouse was injected into gpl20 N with the same way, the last one was gpl20 N/IFN- Y group in which each mouse was injected into gpl20 N/IFN- Y with the same way. Every mouse was injected three times firstly, each time was intervalled 3 days. 2 weeks later 5 mouses of per group would be killed through drawing blood from eyeframe. The blood would be used to do such 5 experiments as IFN- Y activity, Ig antibody(IgG/IgM), CTL activity, gpl20-induced spleen lymphocyte cell enlargement and gpl20-induced spleen cell CKs expression of Thl and Th2 type.( IFN-Y ,IL-2,IL-4,IL-10)o The rest mousse would be injected respectively into PBS ,gpl20 and gpl20N/IFN-Y after 4 weeks. 3 days later, they would all be killed through the same way, and then the blood would be tested for Ig antibody and CKs expression (the same as above). Results1. pet44b-gpl20 N and pet44b-gpl20N/IFN- Y plasmid were verified by double digesting and DNA sequence analysis. The results were consistent with those expected. Therefore the above two kinds of recombinant plasmids were successfully constructed.2. Using the pured gpl20 N protein and gp!20 N/IFN- Y protein stimulated theBALB/c mousse respectively, the results were as following:1) IFN- Y played the role of immune adjuvant and increased the excrete level of specific antibody. The excrete level of IgG in the second immune reaction was 6-8 times higher than the first immune reaction. The antibody levels of the fusion protein and gpl20 N protein were higher than those of the control group, there were significant differences. IgM excrete levels of fusion protein and gpl20 protein in the first time immune reaction were higher than those of the control group, there were significant differences. But in the second time immune reaction, the excrete levels of IgM in the above two groups were without apparent rise.2) In the CTL against gpl20, IFN- Y played an apparent role of being as immune adjuvant. The CTL activity of gpl20N/IFN-y was significantly higher than the gpl20N, much higher than control. The CTL% by LDH released method was respective 0.48 + 0.12> 0.22 + 0.09 and 0.09±0.01, PO.05.3) In the gpl20-induced spleen lymphocyte cell enlargement, IFN- y also played an apparent role of being as immune adjuvant. The OD560 of fusion was higher than that gpl20 N, the one of gpl20 N was higher than that of control. They were respective 0.93 + 0.06> 0.68 + 0.06 and 0.40 + 0.05,P<0.05.4) In the CKs expression, no matter the result from serum or from supernatants, IFN- Y, as immune adjuvant, strengthed the cell immune reaction, and did not impact on the liquid immune reaction. That was, IFN- Y stimulated and induced the body to increase the Thl type CKs excrete level. And the level of Thl type CKs of fusion group was higher than that of gpl20 N group, and the latter was higher than that of control. P<0.05. while the excrete level of Th2 type CKs among the three groups were not significantly different.P>0.05.Conclusions1. Successfully constructed the prokaryotic expression plasmid pet44b-gpl20 N and pet44b-gpl20N/IFN-Y.2. Successfully expressed the protein of gpl20 N and gpl20 N/IFN- Y from DE3 cells.3. As immune adjuvant, IFN-Y stimulated and induced the mouse to increase the antibody expression, strengthened the spleen lymphocyte cell enlargement and Thl type CKs expression, that was, strengthened the cell immune reaction.