Study On High Expression And Purification,Anti Melanoma Activity By The Recombinant Toxin Targeted MC1R And Related Targeting Signal Analysis

Melanoma is a highly malignant tumor with early metastasis,growth rapidly and poor prognosis developed from the melanocytes.Melanoma is the main cause of death related to skin cancer in developed countries.According to statistics data,themelanoma incidencerateisincreasing by around 3⁸ percent per year in the world.Recent decades,China has showing a obvious increased incidence of melanoma prevalent trend and it is estimated that there are 20,000 new cases everyyear,which is also one of the life health diseases that seriously affect the people of our country.Therefore,early diagnosis and targeted therapy for melanoma has become particularly important.At present,the best treatment for melanoma is surgery,but there is still a risk of recrudesce and Tumor migration.Nevertheless,as most patients were advanced melanoma when they were admitted to hospital for diagnosis,the patients do not haveany chances for operation.Although some adjuvant treatment,such as chemotherapy,radiotherapy and immunotherapy,can be used in the treatment of melanoma,melanoma is not sensitive to chemotherapy and radiotherapy resulted in no effective treatment to this disease.Biological immunization therapy,such as melanoma vaccine,is still under the research and development and their long-term efficacy requires further investigation.In recent years,targeted therapy opens a new way in tumor treatment and it is expected to become one of the main direction in melanoma therapy.The diagnosis methods of melanoma are comprehensive clinical examination,biopsy,imaging examination and laboratory test.However,there is still no specific tumor marker for melanoma,which have limitations in improving the accurate diagnosis.As targeted drug intervention should be individualized case by case,they are required to be screened by targeting diagnosis methods in clinical trials and clinical treatment.Tumor marker is significant for establishment of targeting diagnosis methods.With the combination of melanocyte-stimulating hormone（a-MSH）and codon-optimizedpseudomonas exotoxin（PE）,we construct a recombinant toxin targeted Melanocortin 1 Receptor（MC1R）with high expression and specificity and purify it.In order to set up a foundation for future clinical trials and provide guidance for designing anindividualized treatmentfor melanoma,we analyzed the druggability evaluation of recombinant toxin by cytotoxicity test and animal model test and studied the relationship between MC1R expression level in different cell lines and validity of recombinant toxin.The relationship between the expression of Transcription Elongation Factor A（SII）-Like 7（TCEAL7）and miRNA-758 was predicted by Bioinformatics and then we explored the function of miR-758 on the expression of TCEAL7 and its role in melanoma progression.miR-758/TCEAL7 may serve as a new marker for the diagnosis of melanoma and therapeutic target.To obtain a new recombinant toxin with higher expression and low immunogenicity,we truncated the amino acid sequence of the PE40 preserved in our laboratory and changed the carboxyl terminal REDLK into KDEL by mutagenesis.Then,by the aid of the bioinformatics software and the Escherichia coli biased codon,PE38_KDEL was built.PE38_KDEL was combined with a-MSH through an optimized flexible linker and then a-MSH-PE38_KDEL gene was obtained.Then the gene was linked to the expression vectors pET-30a,to construct a recombinant plasmid pET-30a/a-MSH-PE38_KDEL.The plasmid pET-30a/a-MSH-PE38_KDELwas translated into E.coli,and selected positive clones by kanamycin resistance,furtherapproved by PCR and sequencing.Theverified positive clones were inoculated in Kanresistance LB medium and IPTG was used to induce target protein a-MSH-PE38_KDEL expression,and identified with electrophoresis and immunoblotting.The gel image analysis software was used to the analysis on expression level of target protein a-MSH-PE38_KDEL,which took 35%of the whole bacteria protein.The Western blot was used to analyze the soluble or inclusion body expression of recombinant protein a-MSH-PE38_KDEL after bacterial cells ultrasonic disruption.The soluble expression of recombinant protein a-MSH-PE38_KDEL was purified by crude purification and precision purification.Firstly,most of the impurities protein in a-MSH-PE38_KDEL was removed by saturated ammonium sulfate precipitation.Subsequently,it was purified by hydrophobic chromatography and purified by ion exchange chromatography.Finally,the gel filtration chromatography is used for desalination.The salt precipitation and chromatographic situation of the saturated ammonium sulfate are optimized for the whole purification process.The inclusion body expression of recombinant protein a-MSH-PE38_KDEL was purified by denaturation,renaturation and affinity chromatography from inclusion body.The purified results showed that the purity of final products of two means are both quite pure,meeting the needs of the future test.Cytotoxicity test was utilized to analyze the binding of a-MSH-PE38_KDEL with its receptor.Whether a-MSH-PE38_KDEL could induce apoptosis was identified by flow cytometry.In this study,the high expression of MC1R gene in human melanoma cells and murine melanoma B16-F10 cells was measured at the cell level,comparing them with MDA-MB-231,BGC-823,GBC-SD,LO2 cells,whose MC1R expressions are low,in order to confirm its targeted killing effect.The results showed that a-MSH-PE38_KDEL could target cell surface by identifying MC1R,then killed the target cells;one of the main mechanisms for a-MSH-PE38_KDEL showed cytoactivity was inducing apoptosis.Melanoma A375 and B16-F10 cell inhibition rate of a-MSH-PE38_KDEL was over 93%,and it appeared to have no inhibitory effect on human normal LO2 cells.In order to further understand the anti-tumor activity of a-MSH-PE38_KDELin vivo,a melanoma BALB/c mouse model was established and treated by a-MSH-PE38_KDEL to observe its antitumor effects,influence on side effects and obtain safety evaluation.Melanoma mouse models were divided into three treatment groups:high,medium and low doses.All three groups could inhibit tumor growth and metastasis.The high dose group was superior to the other groups,and targeted cells were obviously killedby recombinant toxin,and the tumor disappearance rate reached 58%.After long-term（50days）high dose treatment,tumor disappeared in 4 cases and obviously reduced in 3 cases.Moreover the pathological results of sliced tissues showed that a-MSH-PE38_KDELDEL had no significant side effects on BALB/c mice.The significant differences in MC1R expression level between tumor cells and normal cells indicate that it used as a testing target or targeting therapy,we established a method to detect MC1R.The real-time fluorescence quantitativePCRand Western-blotting methods were used respectively to detect MC1R in a variety of cells qualitatively,we analyzed the relationship between MC1R expression level and a-MSH-PE38_KDEL validity.The results showed that MC1R is highly expressed in B16-F10 cells,however,the amount of MC1R expressed in HT29 and eca-109 cells is3 to 5 times higher than that expressed in B16-F10 and MC1R expression level in HCT-116 cells is similar to B16-F10.The results of the fluorescence quantitative PCR method are basically the same as that detected by the Western-blot method.TCEAL7 has been observed in many kinds of cancer,include melanoma,but the function of it on the development of melanoma is still unclear.The low expression of TCEAL7 in melanoma cell lines and clinical tissues was detectd by RT-PCR and Western blot.Up-regulated the expression of TCEAL7 in A375 and WM-115 cells by infecting lentivirus and down-regulated the expression of TCEAL7 by transfecting siRNA-TCEAL7.CCK-8 assay was used to analyze the effect of TCEAL7on melanoma cell growth and flow cytometry was used to test the role of TCEAL7 on melanoma cell apoptosis.The results displayed that inhibition the expression of TCEAL7 inhibited cell apoptosis and promoted cell proliferation in WM-115and A375 cells.The malignant melanoma A375 and WM-115 cell after transfection and infection were re-suspended and then injected into 4-week-old female BALB/c nude mice（1×10⁷cells）.The results displayed that up-regulation of TCEAL7 repressed the tumorigenesis of WM-115 and A375 cells and down-regulation of it promoted the tumorigenesis.The bioinformatics analyses displayed that the co-regulators of TCEAL7 was miR-758.The results displayed that the pruduce level of miR-758 was raised and the expression of TCEAL7 was inhibited obviously when A375 and WM-115 cells were transfected mimics-miR-758.The biluciferase report gene results showed that the transcriptional activity of TCEAL7 were repressed when up-regulation of miR-758 in melanoma cells.CCK-8 results demonstrated that cell proliferation was increased when WM-115and A375 cells were transfected mimics-miR-758 and this function was impaired when over-expressed TCEAL7 on the base of mimics-miR-758.Moreover,cell apoptosis was inhibited when WM-115 and A375 cells were transfected mimics-miR-758 and this effect was impaired when treated WM-115 and A375 cells with Lentiv-TCEAL7 on the base of mimics-miR-758.These results indicated that TCEAL7 as a suppressor in melanoma oncogenesis and miR-758 served as an oncogene in melanoma progression through inhibiting TCEAL7 expression.To summarise,the expression level of the target protein of a-MSH-PE38_KDELDEL strain increased from 10%to 35%⁴0%of total somatic proteinthrough PE40 codon optimization.Laboratory scale purification procedures of the recombinant protein was basically well established and the final purity of products reached 95%.The tumor targeting cell lines and dosage of the recombinant toxin was determined.The research on therapy of the melanoma animal models confirmed anti-tumor potency of a-MSH-PE38_KDEL.It may be a promising targeted drug for melanoma treatment as it can effectively eliminate melanoma tumors and clinical observation,body weight and histomorphology tests of BALB/c mouse models intargetingtreatmentgroup were all in normal range.Several new tumor targeting cell for recombinant toxin was found in cell targeting screening,include HT29 and eca-109 cell lines,and the target killing effect is significantly stronger than the melanoma cell B16-F10.Through the evaluation of the cells and tissues of melanoma,we also confirmed miR-758promoted the progression of melanoma through down-regulating TCEAL7 expression in melanoma cells,which might provide a new diagnostic marker and therapeutic target for melanoma.Forallthat,it is hoped that the results of this experiment can provide some reference for the screening of clinical cases and the individualized treatment of melanoma in the future clinical trials.