| HIV is the unique pathogen of AIDS. There are two existing forms of HIV virus in AIDS patients: both in the HIV infected CD4+ cells and blood circulation as free HIV virus. AIDS patients would be cured if the two forms of HIV virus were cleared away completely. Historically, there was an engineered CD4-PE40 (Domain II-III of Pseudomonas aeruginosa), which was used to reach this aim. However, due to the strong immunogenicity of PE40 as a heterologous protein for human, this attempt was concluded to be a failure finally.In this project, we tried to construct a series of fusion proteins containing a binding domain that targets the gp120 and a cytotoxic domain that actively kills the HIV/SIV infected cells. Both of them are the homologous protein for human so that the immunogenicity of recombinant protein was minimized. Moreover, each domain would keep the original three-dimensional structure, which avoid the producing of antibody against the fusion protein due to being recognized wrongly as the foreign protein by human immune system. Gp120 were expressed only on the surface of the HIV/SIV infected cells or free virus. The normal cells wouldn't express such a kind of envelope protein. On one hand, the fusion protein would enter the HIV/SIV infected cells through gp120-mediated internalization and cytotoxic domain would also kill them selectively. On the other hand, the binding domain of fusion protein would bind the free virus and block the virus spreading so that protect the normal cells finally. In a word, as a kind of bifunctional polypeptide, the series of fusion proteins enable the HIV/SIV infected cells and the free virus to be cleared up efficiently.This project has been done with SIV as its subject in terms of the parallel relationship of the HIV and SIV in biological behavior. There are three parts in this dissertation.Part oneThe study of TNFαm1 type targeting fusion proteinTNFαm1 was a kind of highly active TNFa mutant, in which the three amino acid( Gly, Thr and Arg)and the two amino acid (Gly, Pro )were added front and behind the first amino acid Val at N termimal of TNFαrespectively.Based on the E. coli. coden usage, we obtained the coding sequence of bindingdomain of the fusion protein and constructed these 4 TNFαm1 types of fusion protein.1) Fusion protein CD4V1TNFαm1: As the key binding domain of CD4 with gp120, the coding sequence of V1 domain of CD4 containing 100 amino acid named CD4V1 were fused to the N terminal of TNFαm1 to construct the fusion protein CD4V1TNFαm1.2) Fusion protein RNTNFαm1: As the key binding domain of CCR5 with gp120, the N terminal region of CCR5 containing 30 amino acids named RN were fused to the N terminal of TNFαm1 to construct the fusion protein RNTNFαm1.3) Fusion protein XETNFαm1: As the key binding domain of CXCR4 with gp 120, the second extracellular loop of CXCR4 containing 27 amino acids named XE were fused to the N terminal of TNFαm1 to construct the fusion protein XETNFαm1.4) Fusion protein XERNTNFαm1: It is reported that R5 HIV strains would convert to X4 viruses in vivo typically occurs after several years of infection. We used the XERN as the target domain in order to get the "bi-tropic" molecular to exert function at different disease progression of AIDS.The expression recombinants were transformed into expression host strains DH5αor BL21 to get the engineered strains pCW-XETNFαm 1/DH5α,pCW-CD4V1TNFαm1/DH5α,pCW-XERNTNFαm1/DH5αas well as pET30-RNTNFαm1/BL21. Laser density scanning showed that the expression level of the 4 targeting fusion proteins, XETNFαm1, CD4V1TNFαm1, XERNTNFαm1 as well as RNTNFαm1 could reach to 27.4%,10.8%,14.2% and 15.6% of total cell protein respectively. The fusion protein were isolated and purified from SDS-PAGE gel. After that, biological activities assay at cell level in vitro of the fusion proteins has been investigated. Different dosage fusion proteins were added to 2.0×105/ml CEMx-174 cells infected by SIV respectively. The killing ratess of the fusion protein in the observed dose range(8,16,32,64,128μg/ml) were as follows:1) The killing rates of XETNFαm1 were 17.3%, 2.9%, 8.7%, 45.4%, 54.9% respectively;2) the killing rates of CD4V1TNFαm1were 9.5%, 26.7%, 32.0%, 44.3%, 52.4% respectively;3) the killing rates of XERNTNFαm1were 13.7%, 24.5%, 31.9%, 37.1%, 45.6% respectively;4) the killing rates of RNTNFαm1were 16.2%, 20.3%, 28.2%, 31.7%, 33.7% respectively.Moreover, MTT test indicated TNFαalone exerted no killing function on SIV-infected cells while the four fusion protein exerted no killing function on normal CEMx-174 cell lines(P＞0.20). The results showed that TNFαm1 type targeting fusion protein could selectively kill cells expressing gp120 through the gp120 mediated cell internalization while it could be safe to the normal cells as a result of absence of gp 120. The results were consistent with the original design.In addition, results of virus titer assays showed that in the observed dose range(8,16,32,64,128μg/ml) were as follows:1) XETNFαm1: virus titers from original 1: 5120 drop to 1: 640, 1: 320, 1: 160, 1: 40, 1: 20 respectively;2) CD4V1TNFαm1: virus titers from original 1: 5120 drop to 1: 1280, 1: 640, 1: 80, 1: 40, 1: 10 respectively;3) XERNTNFαm1: virus titers from original 1: 5120 drop to 1: 1280, 1: 640, 1: 320, 1: 160, 1: 40 respectively;4) RNTNFαm1: virus titers from original 1: 5120 drop to 1: 2560, 1: 640, 1: 160, 1: 40, 1: 40 respectively.Moreover, It was showed that there was a good dosage-effect correlation between the dosage and the decline of virus titer. (2)On the other hand, The results of neutralization assay showed that the OD value of experiment group was significantly higher than that of the control group (p＜0.05). The neutralizing effects of the fusion proteins in the observed dose range(2, 4, 8, 16, 32μg/ml) were as follows:1) The protecting rates of XETNFαm1 were 12.7%, 15.7%, 26.6%, 33.5%, 40.2% respectively;2) the protecting rates of CD4V1TNFαm1were 8.7%, 13.2%, 20.4%, 27.7%, 34.6% respectively;3) the protecting rates of XERNTNFαm1were 15.1%, 18.8%, 21.2%, 32.7%, 38.5% respectively;4) the protecting rates of RNTNFαm1were 13.3%, 18.6%, 19.5%, 24.3%, 31.8% respectively.In addition, results of virus assays test showed that in the observed dose range((2, 4, 8, 16, 32μg/ml) were as follows,1) XETNFαm1: virus titers from original 1: 5120 drop to 1: 1280, 1: 320, 1: 160, 1: 40, 1: 20 respectively;2) CD4V1TNFαm1: the virus titer from original 1: 5120 drop to 1: 1280, 1: 320, 1: 80, 1: 40, 1: 20;3) XERNTNFαm1: the virus titer from original 1: 5120 drop to 1: 1280, 1: 640, 1: 160, 1: 160, 1: 40 respectively;4) RNTNFαm1: the virus titer from original 1: 5120 drop to 1: 2560, 1: 640, 1: 160, 1: 40, 1: 40 respectively.Similarly, it was also showed that there was a good dosage-effect correlation between the dosage and the decline of virus titer.Part twoThe study of TNFαm2 type targeting fusion proteinTNFαm2 was an other kind of highly active TNFa mutant, in which 7 N-terminal amino-acids were deleted and Pro8Ser9Aspl 0 was replaced by ArgLysArg. Compared with wild-type recombinant TNFα, TNFαm2 had a 7-fold higher cytotoxic in vitro. Moreover, the toxicity of TNFαm2 was 18 times lower than that of wild-type TNFα. TNFαm2 has already been extended to clinical utilization. The Env-binding moieties, CD4V1, XE and XERN were fuseed to the N terminal of TNFαm1 respectively to construct a series of targeting fusion protein: CD4V1 TNFαm2, XE TNFαm2 and XERN TNFαm2. The expression recombinants were transformed into expression host strains DH5αand BL21 to get the engineered strains pCW-XETNFαm2/DH5α,pET30a-CD4V1TNFαm2 and pET30a-XERNTNFαm2/ BL21. Laser density scanning indicated that the expression level of the three targeting fusion proteins, XETNFαm2, CD4V1TNFαm2 and XERNTNFαm2 could reach to 38.6%, 21.2%, 24.5% of total cell protein, respectively. CD4V1TNFαm2 and XERNTNFαm2 were isolated and purified from SDS-PAGE gel. XETNFαm2 was isolated and purified by RHPLC of AKTA. Amino acid sequencing of N-terminal of XETNFαm2 was MNVSEADDRYIXDRF. LC-MS/MS identified the XETNFαm2 was a kind of tumor necrosis factor alpha[homo sapiens] and revealed the molecular weight was 17.4KD and Isolectric Point was 8.6. Followed that, the cell activity experiment of the fusion proteins on 2.0×105/ml CEMx-174 cell lines has been investigated.(1)The results showed that the OD value of experiment group was significantly lower than that of the control group (p＜0.05). Meanwhile, the killing ratess of the three protein were as follows:1) in the observed dose range(1,2,4,8,16μg/ml), the killing rates of XETNFαm2 were 21.0%, 30.0%, 37.6%, 49.8%, 65.3% respectively.2) In the observed dose range(2,4,8,16,32μg/ml), the killing rates of CD4V1TNFαm2were26.9%, 31.6%, 40.0%, 44.7%, 53.6% respectively;3) the killing rates of XERNTNFαm2were 18.7%, 29.6%, 33.5%, 40.2%, 49.7% respectively.Moreover, MTT test indicated that both TNFαalone have no killing function on SIV-infected cells and three fusion protein exert no killing function on normal CEMx-174 cell lines(P＞0.20). The results also showed that TNFαm2 type targeting fusion protein could selectively kill cells expressing gp 120 through the gp120 mediated cell internalization while it could be safe to the normal cells without gp120. The results were consistent with the original design.In addition, results of virus titer test showed that,1) in the observed dose range(1,2,4,8,16μg/ml), XETNFαm2 made the virus titer drop to 1: 1280, 1: 640, 1: 80, 1: 40, 1: 20 from original 1: 5120 respectively.2) In the observed dose range(2,4,8,16,32μg/ml), from original 1: 5120, CD4V1TNFαm2 made the virus titer drop to 1: 1280, 1: 640, 1: 320, 1: 40, 1: 40 respectively;3) In the observed dose range(2,4,8,16,32μg/ml), from original 1: 5120, XERNTNFαm2 made the virus titer drop to 1: 2560, 1: 640, 1: 160, 1: 40, 1: 20 respectively.Moreover, It was also showed that there was a good dosage-effect correlation between the dosage and the decline of virus titer.(2)On the other hand, The neutralizing effects of the fusion proteins showed that the OD value of experiment group was significantly higher than that of the control group (p＜0.05). The neutralizing effects of the fusion protein were as follows:1) In the observed dose range(1,2,4,8,16,32μg/ml), the protecting rates of XETNFαm2 were 13.6%, 18.9%, 29.1%, 38.0%, 50.4%respectively.2) In the observed dose range(2,4,8,16,32μg/ml), the protecting rates of CD4V1TNFαm2 were 8.6%, 15.7%, 23.6%, 30.5%, 42.0% respectively;3) the protecting rates of XERNTNFαm2 were 7.3%, 21.3%, 28.6%, 34.7%, 39.6% respectively.At the same time, results of virus titer test showed that at different dose range,1) from original 1: 5120, XETNFαm2 made the virus titer drop to 1: 1280, 1: 640, 1: 160, 1: 40, 1: 20 respectively;2) CD4V1TNFαm2 made the virus titer drop to 1: 1280, 1: 320, 1: 80, 1: 40, 1: 20 respectively;3) XERNTNFαm2 made the virus titer drop to 1: 2560, 1: 1280, 1: 320, 1: 160, 1: 20 respectively.Similarly, it was also showed that there was a good dosage-effect correlation between the dosage and the decline of virus titer.In addition, the apoptosis ratess of different group determined by FACS were as follows: In killing assays, control group was 21.2%; at the dose of 16μg/ml, XETNFαm2 treated group: 47.1%; TNFαtreated group: 20.8%. In neutralizing assays, the control group: 20.3%, XETNFαm2 treated group: 11.3%. The results lend the support to the opinion that these targeting fusion proteins could selectively induce the apoptosis of HIV/SIV infected cells and protect the normal cells.Part threeThe study of HP type targeting fusion proteinIn addition to the above researches, another study has been carried out. The active regions of human perforin containing 104 amino acid of N terminal was fused to N terminal of XE to construct the fusion protein HP104XE. Perforin can perforates target membranes by forming transmembrane pores, such that target cells lose membrane integrity and appear to die of colloid osmotic lysis. The two expression recombinants were transformed into JM109 and BL21 to get the engineered strains pGEX4T1-P312XE/ JM 109 and pET32a-P312XE/BL21. Laser density scanning of SDS-PAGE revealed the expression level of the two fusion protein could reach to 10.5 % and 37.6% respectively.The general conclusion that can be drawn from above studies is that, these kinds of fusion proteins would bind onto and selectively kill HIV/SIV infected cells expressing gp120 protein on their surface. At the same time, they could also bind the free virus and thereby block viral infectivity, replication and syncytium formation. Furthermore, these results supported the potential therapeutic utility in AIDS therapy. Besides AIDS, it is very likely to be extended for the efficient therapy of other disease such as cancer and autoimmunogical disease.
|
PDF Full Text Request