| It is known that thousands of naturally occurring toxin proteins possess anticancer activity. But few of them were applied in clinical research, and none was successful. The obstacles exist in two aspects, nonspecificity and immunogenicity. The toxins administrated in vein leads to the production of antibody against itself, thus the neutraliztion response later limits the cure effect of toxin, even leads to the invalidation. The targeting fusion protein with toxin was constructed appeared in last century consist of ligand to receptors on tumor cells and function domains of toxins. They are constructed with the technique of DNA recombination, and expressed fused. Targeting toxin could limit the toxicity in cells with corresponding receptors, but the toxicity to normal cells with the same character could not be avoided. The immunogenicity could not be eliminated either.Aiming to the obstacles against application of toxins in tumor therapy, we proposed a design, to avoid not only the immunogenicity but also the toxic side effect. In this project the expression recombinants with coding sequence of PEIIImut toxin protein. A kind of fusion gene was done and expressed. Its fusion protein contained GnRH as the targeting molecule and partial sequence of Son as DNA binding polypeptide. Toxin expression recombinant could be packed by the SON domain of fusion protein, and was led to the one kind of cancer cells with the guidance of GnRH. In cells with GnRH receptors, toxin protein were expressed, and cancer cells were killed by cytotoxic function. On the basis of the hypoxia specialty of tumor cells, hypoxia response element was applied to regulate the expression of toxin gene. Therefore the normal cells with GnRH receptors could not be killed since they are aerobic. Thus, both the immunogenicity and the toxic side effect would be avoided in this design.The coding sequence of GnRH was obtained by PCR, and so was the 72 amino acids of Son. The fusion sequenc of GnRHSON was inserted in pPIC9K of pichia pastoris expression system. Linearized recombinant was transformed into KM71 with use of electroporation. Using G418 growth inhibition for screening, some strains showed high expression of GnRHSON. The yields were about 1mg/L, and the target protein secreted into the culture medium reached 58.5% of total supernatant proteins. The strain giving the highest expression level was inoculated and induced to harvest the target fusion protein. Then it was purified by precipitation of ammonium sulfate, dialysis, concentration in succeeding procedures. To enhance the expression level, the expression system was switched to E. coli, and the codens of fusion protein were changed to the ones E. coli preferring. To improve the transfering and expression efficiecy in vivo, one kind of nuclear locating sequence, TAP, was inserted between GnRH and SON. Then the coding sequence of GnRH-TAP-SON was cloned into several vectors, and expression was obtained by recombinant pCW-GnRH-TAP-SON, in which target protein was fused to Ref, a P1 phage gene. Thrombin recognizing sites were added to the N terminal of target sequence to make the cleavage possible. But the fragments caused after cleavage were too resembling to separate. They got the same electrical property, and the molecular mass were closed nearly. So another recombinant was constructed, pCW- GnRH-TNF87-TAP-SON, and the protein was expressed as inclusion body. The purification procedure included reverse phase column, cation exchange column and hydrophobic interaction column. The purity of the target protein was tested by silver staining after SDS- PAGE electrophoresis, and only one bind appeared.The toxin gene used in this project was the mutant coding sequence of the third domain in PE. The last four amino acids were replaced as KDEL to enhance the toxicity. Four-copy HRE was inserted into the upstream of CMV promoter, and the promoter was also deleted to be its mini form. To enhance the activity of the promoter, intronⅡin globin of rabbit was inserted between miniCMV and toxin gene. The toxin expression recombinant was purified by chromatography, after precipitated by CaCl2, first using the DEAE anion ion exchange column, second using Resource Q column. The supercoiled form accounted to be 90％at last. Protein couldn't be detected by SDS-PAGE electrophoresis and BCA kit.The solution of target protein and DNA was mixed at different ratios (1: 1, 2: 1, 4: 1, 8: 1). Electrophoresis of agarose gel showed 1: 1 was the best. DNA-protein complex was stored at 4℃one month, electrophoresis result showed DNA was almost detained in sample hole. It illustrated complex was stored steadily at 4℃.The recombinant plasmids coding green fluorescence protein(GFP) and luciferase reporter gene were tranfected by lipofectin and purified fusion protein as nonviral vector, respectively. Green fluorescence protein and luciferase were both expressed. The results showed that fusion protein as nonviral vector and protein-DNA complex transfected tumor cells was effective.Cell lines expressed GnRH receptor HEK293(human embryonic kidney, normal cell line), HepG2 (Hepatocarcinoma), Hela(cervix carcinoma) and MCF-7(mammary cancer), and cell line 2BS (human embryonic lung) without GnRH receptor expression were used in the targeting killing experiment. Under aerobic condition, pGL3-SV40-PEⅢ/protein GnRH-TNF87-TAP- SON complex could kill all of the receptor positive cells strongly, but could not kill the negative cells such as 2BS. When dose of complex was 8 ug/ml, the killing rates were 72.9％, 57.9％, 54.1％and 51.7％corresponding to HEK293, HepG2, Hela and MCF-7. The results suggested that the fusion protein GnRH-TNF87-TAP-SON could lead the toxin recombinant into all GnRH receptor positive cells including of normal GnRH receptor positive cells. Thus, the project maybe lead side effect and was not consummate. Under the same condition, nonviral vector and pGL3-4HRE-miniCMV-PEⅢcomplex showed no killing effect on HEK293. So the toxic side effect could be avoided by the control of GnRH and HRE- miniCMV.Under the hypoxia condition, using nonviral vector protein GnRH-TNF87-TAP-SON and pGL3-SV40-PEⅢat the dose of 8 ug/ml could kill the positive GnRH receptor cells such as HepG2, Hela and MCF-7, and the killing rate was 61.3％,60.1％and 53.9％, respectively. It further showed SV40 promoter was not desirable. Function gene controllable expression was designed, then accurate targeting killing could be come true. Nonviral vector protein GnRH- TNF87-TAP-SON and pGL3-HRE4-miniCMV-PEⅢat the dose of 8 ug/ml could kill the positive GnRH receptor cells such as HepG2, Hela and MCF-7, and the killing rate was 57.1％, 56.9％, 41.7％, respectively.Under the hypoxia condition, nonviral vector protein GnRH-TNF87-TAP-SON and pGL3-HRE4-miniCMV-PEⅢcould lead cell apoptosis through flow cytometer analysis.Fusion protein designed in this project could mediate toxin gene into the GnRH-R positive cells, the toxin protein expressed and killed the cells. With the control by HRE- miniCMV, the expression of toxin was limited only in hypoxia GnRH-R positive tumor cells, the normal cells with GnRH-R could not express the toxin protein due to its aerobic characteristics, and could not be killed. Thus, normal cells without GnRH-R and the normal cells with GnRH-R could not be killed. Because the toxin gene could not be expressed in normal aerobic cells. The bi-switch design utilized both relationship of receptor and ligand and hypoxia condition as controllable gene expression, then the gene therapy design of accurate targeting killing tumor cells could be come true. Toxin gene used in the controllable expression design was first.The design demands individual diagnosis and individual treatment. Because the complex drugs only fitted to hypoxia tumor cells with positive GnRH receptor, such as Hepatocarcinoma, mammary cancer, colorectal tumor, pancreatic cancer, ovary tumor, prostate carcinoma and pituitary tumor. It is the preliminary exploration of Medicine in 21 century, characterized as "individual diagnosis and individual treatment". |
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