| HIV is the unique pathogen of AIDS. There are two existing forms of HIV virus in AIDS patients: one existing in the infected CD4+ cells and another in the blood circulation as free HIV virus. AIDS patients can be cured if such two forms of HIV virus are cleared away. Historically, there was a engineered design regarding CD4-PE40 (Domain II-III of Pseudomonas aeruginosa, exotoxin) which was used to reach this aim [1-15]. But due to the very strong immunogenicity of CD4-PE40 protein resulted from engineered E.coli and PE40 as heteroprotein , this attempt leaded to failure. Yet as first-generation IT (immunotoxin) targeted to gp120, CD4-PE40 (chimeric immunotoxin using CD4 and enzymatic domains of Pseudomonas exotoxin A), showed limited promise in initial clinical testing, highlighting the need for improvement. [16]. The new strategy was utilized to both exert function of Pseudomonas exotoxin A and avoid side effect ,such as immunogenicity of heteroprotein . Detailed strategy was as follows: Fusion proteins composed of receptor and coreceptor and DNA-binding Protein were used as noviral carrier which can combine with PEIIImut expression recombinant to form protein-DNA complexes and transect PEIIImut DNA recombinant into cells infected by HIV. There are two kinds of toxin gene recombinant. One is unrestricted recombinant pCMV-PEIIImut which uses CMV as promotor. Another is restricted recombinant pYL-PEIIImut and pDYP-1 (Domain III mutant of Pseudomonas aeruginosa exotoxin) which uses HIV-5'LTR as promotor. PEIIImut can be activated by TAR protein which exists in cells infected by HIV virus because promotor of PEIIImut DNA recombinant is 5'LTR. PEIII mutant protein expression can kill the cells infected by HIV in a targeting way. When PEIIImut DNA recombinants were mis-transfected into normal cells, they can't be expressed due to TAT protein absence in normal cells. The safety can be ensured by dual-key control. In the meantime, the N-terminal part of the fusion protein will also combine the free HIV virus existing in the blood stream, making it lose infectivity because of neutralization. Thirdly, this complexes can prevent cell-cell fusion by binding to gp120 on the surface of infected cells. Such a design could avoid the immunogenicity and cell toxicity to kill any other kind of unifected cells.The reported key binding regions of CD4, CXCR4 and CCR5 used as receptor and coreceptor by HIV are V1 domain of CD4 containing 100 amino acid, the second extracellular loop of CXCR4 containing 27 amino acids and the N terminal region of CCR5 containing 30 amino acids as targeting protein. DNA-binding protein, SON spanning 72 amino acids which is rich in the positive amino acids, was chosen. The targeting protein and the DNA-binding protein were engineered to be fusion proteins, shch as XRSON(composed of XE-RN-SON), CD4Vl-RN-SON(composed of CD4V1-RN-SON), CD4V1-XE-RN-SON(composed of CD4V1-XE-RN-SON), CD4V1SON(composed of CD4V1-SON). N-terminal of Fusion proteins was the receptor or coreceptors of HIV, such as CD4, CXCR4 and CCR5 that can bind Env gp120 molecule on the surface of infected cells by HIV and the primary free HIV virus. C-terminal was DNA-binding peptide from SON gene which is rich in positive amino acids and binds PEⅢmut DNA recombinant via charge interaction. N-terminal can carry the whole DNA-protein complex into cells infected by HIV. PEⅢmut DNA recombinant plasmid can express PEⅢprotein which can kill the cells infected by HIV. In the meantime, receptor or coreceptors of HIV can bind the Env gp 120 of free HIV virus in the blood stream, making virus lose infectivity by neutralization.Coding fusion genes were inserted into expression vector, pCW111 whose promoter is controlled by temperature. Only XRSON and CD4V1 SON were expressed well. After the induction and harvest of the engineered bacteria, the fusion proteins were isolated and purified by AKTA. The expression rate of fusion protein XRSON was 13％. The expression rate of CD4V1SON was 35％. Physiochemical property of XRS was identified. High-resolution MALD-TOF evaluation revealed that the molecular weight of XRS was 15703.44.15. Amino acid sequencing of N-terminal of XRSON was MNVSEADDRYIXDRE Capillary electrophoresis revealed that the Isolectric Point of XRS was 10.99. The constitutes of amino acid of XRS protein was the same as design. Purity of XRS revealed by HPLC was 93.18％.Two kinds of recombinants (pCMV-PEⅢmut and pDYP-1,) expressing the mutant format of domainⅢof Pseudomonas aeruginosa exotoxin were used to kill cells infected by HIV/SIV. Their promoters are CMV promoter as unrestricted recombinant and HIV-1 5'LTR as restricted recombinant, respectively. The tat protein expression in infected cells by HIV can activate 5'LTR promoter, so that the toxin gene PEⅢmut will be especially expressed in cells infected, Though pDYP-1, recombinants was sent into in normal cells, PEⅢmut protein will not be expressed without the existence of tat protein. Cells will not be killed accordingly. To enhance tat expression level in infected cells, 5'LTR-Intron-PEⅢmut recombinant pDYP-1 were constructed by inserting IntronⅡsequence under stream of 5'LTR sequence. DNA-protein complexes as drugs were formed by mixing fusion protein XRS with recombinants (pCMV-PEⅢand pDYP-1, respectively).DNA-protein complexes were formed, such as CD4V1SON/pDYP-1,XRSON/pDYP-1 which were compared with CD4V1SON/pCMV-PEⅢmut,XRSON/pCMV-PEⅢmut, XRSON+CD4V1SON/pDYP-1,XRSON+CD4V1SON /pCMV-PEⅢmut after toxin DNA recombinants, CD4V1 and XRS were purified. Cell activity experiment had been done with SIV in terms of the similar relationship of the HIV and SIV(simian immunodeficiency virus )in biological behavior. The killing effects of these complexes on the cells infected by SIV were observed respectively and combinationally after CEMX-174 cells was treated with varied doses of complexes(1, 2, 4μg, calculated in DNA). cells in the control group were uninfected by HIV. The neutralizing effects of the complexes on the free SIV were observed respectively and combinationally after CEMX-174 cells was treated with varied doses of complexes(1, 2, 4μg, calculated in DNA) for 48 hours. The normal ceils were mixed with SIV virus as contral. After 7 days, The neutralizing effects of the complexes were significant.The results showed that the killing rates of the complexes in single and combined manner in different dosages(1, 2, 4μg, calculated in DNA) were as follows: the killing rates of XRS/pDYP-1 were 27.91％,37.21％,50.54％, respectively; the killing rate of control cells infected by SIV without complex was 0. Virus titers were 1: 320,1: 320,1: 160, respectively; the titer of control cells infected by SIV without complex was 1: 1280. XRS/pCMV-PEⅢmut were 12.97％,24.36％,43.64％, respectively; the killing rate of control was 0. Virus titers were 1: 320,1: 320,1: 80, respectively; the titer of the control was 1: 1280. XRS+CD4V1/pCMV-PEⅢmut were 12.97％,24.36％,43.64％, respectively; the killing rate of the control was 0. Virus titers were 1: 80,1: 80,1: 40 respectively; the titer of the control was 1: 1280. XRS+CD4V1/pDYP-1 were 18.67％,28.47％and 49.95％, respectively; the killing rate of the control was 0. Virus titers were 1: 80,1: 80,1: 40, respectively; the titer of the control was 1: 1280. The cytotoxicity is dependent on the concentration of complex.On the other hand, the protecting effects of the complexes on the host cells in neutralization assay are as follows: the rates in XRSON (2.3, 4.6, 9.2ug) were 2.4％,10.33％,20.22％, respectively; the protecting rate of the control was zero. Virus titers were 1: 1280,1: 1280,1: 320, respectively; the titer of the control was 1: 1280. XRSON +CD4V1 were-7.4％,4.8％,18.75％, respectively; the protecting rate of the control was zero. Virus titers were 1: 1280,1: 320,1: 80, respectively; the titer of control was 1: 1280. XRSON/pDYP-1 (1, 2, 4μg, calculated in DNA) were-4.3％,3.1％,10.33％, respectively; the protecting rate of control was 0. Virus titers were 1: 1280,1: 640,1: 160, respectively; the titer of control was 1: 1280. XRSON/pCMV-PEⅢmut were 0.5％,2.3％,7.3％, respectively; the protecting rate of control was 0. Virus titers were 1: 320,1: 80,1: 20, respectively; the titer of control was 1: 1280. XRSON+CD4V1/pDYP-1 were-6.4％,15.89％,22.5％, respectively; the protecting rate of the control was 0. Virus titers were 1: 1280,1: 640,1: 80, respectively; the titer of control was 1: 1280. XRSON+CD4V1/pCMV-PEⅢmut were-2.3％,3.1％,21.94％, respectively; Protecting rate of the control was 0. Virus titers were 1: 1280,1: 640,1: 80, respectively; the titer of the control was 1: 1280. The protection is dependent on the concentration of drugs.According to the above results, we concluded that the recombinants can effectively express the target toxin protein which could kill the cells infected by SIV after the fusion proteins effectively mediate the recombinants into the target cells. Moreover, the data showed that the fusion proteins could neutralize the free SIV and lead to the markedly decrease of the infectivity of SIV in different treatment groups. We must emphasize the fact that the killing effect of the complexes didn't absolutely separate from neutralizing effects of the complexes. Some cells can be infected by SIV virus in the neutralizing effect of the complexes. But the neutralizing effect of the complexes is significant. To the contrary, the neutralizing effect of the complexes involves in the killing effect. But the killing effect is significant.To understand the expression of toxin gene in infected cells, two new recombinants was constructed by replacing the toxin gene with the red and green fluorescence protein gene in pCMV-PEⅢmut plasmid and pDYP-1 plasmid. The recombinant and the nonviral vector targeting protein were mixed to produce new complexs. As CEMX-174 cells is so small and tender a kind of suspended cells that the fluorescence protein is difficult to be deserved. One new recombinants was constructed by replacing the toxin gene with the luciferase gene in pCMV-PEⅢmut plasmid and was transfected into 293 cells and CEMX-174 cells. Transfection efficiency is enhanced significantly in 293 cells than in CEMX-174 cells. Those datas elucidated that toxin gene can be activated under the stream of CMV and LTR promoter. In the mean time, FACS showed that PEⅢmut can trigger apoptosis under the stream of CMV when injected into CEMX-174 cells through lipofectAmine TM Reagent. But PEⅢmut can't trigger apoptosis under the stream of LTR promoter when injected into CEMX-174 cells through lipofectAmine TM, resulting from the absence of TAR. When toxin gene recombinants were transfected into normal cells, the effect of killing was significant in the group of pCMV-PEⅢmut plasmid, pDYP-1 plasmid group did't have such effect. Such two Test above showed that the effect of toxin gene under the stream of LTR promoter should be activated by TAR protein. When PEⅢmut DNA recombinants were mis-transfected into normal cells, they can't be expressed due to TAT protein absence in normal cells. The safety can be ensured by dual-key control.Reverse phase column and cation exchange chromatography were selected to purify two kinds of proteins. Proteins purified was natured by dialysis after freeze-dry. Interest protein was obtained at 30％B buffer during Reverse phase column purification. Other proteins were cleaned away. Probational columns involve Gel column, Ion column, affinity column and so on. Some buffers and pH range were used. We establish a successful process to purify XRS and CD4V1SON after 280 times tryout. HPLC mensuration showed that purify of obtained protein is 93.18％. As a result of a large needs of target protein and toxin gene recombinants, they were fermented and purified in large-scale. The process and factors of large-scale fermentation and production were obtained. Large-scale production in industry is possible on the basis of all techniques. Anion exchange chromatographys was selected to purify toxin gene recombinants. The process of rapid purification of toxin gene recombinants was obtained.We are ready for animal test. Effects of protein-DNA-complexes in animal model are exploring further. Such kind of protocols used in this study is a novel strategy for AIDS therapy, and it is very likely to be extended for the efficient therapy of other diseases only if the targeting protein as the nonviral vector could targeted to other suitable receptor on the cell surface of cancer or autoimmune diseases.
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