Expression And Anti-breast Cancer Potency Of Immunotoxin ScFv-Mmut

Breast cancer is one of the most serious malignancies,which is currently a serious threat to women's health.It's a systemic and highly heterogeneous disease.In clinical practice,treating breast cancer with surgery and chemo-radiotherapy can improve patient's condition and extend the survival time,but it will produce serious non-specific damage to the body.At the beginning of the 20 th century,targeting therapy for tumor began to receive attention which can selectively kill tumor cells and decrease the side effects to normal cells.This idea became a hot spot in the field of cancer research.The immunotoxin is a kind of targeting drug that consisted of targeting carrier and toxin molecule(also known as an effective molecule),it is also a fusion protein that is capable of specifically killing tumor cells.Toxin molecules can enter into the tumor cells by the targeting carrier guided which target to tumor cell surface selectively.This method can improve the efficacy while reducing damage to normal tissue cells.Therefore,the choice of special guided carrier and drugable toxins are important for the construction of immunotoxins.Studies have shown that human epidermal growth factor receptor-2(HER-2)is highly expressed in about 25%-30% of breast cancer patients,and is closely related to the growth,metastasis and poor prognosis of breast cancer.Therefore,the study of HER-2 is of great significance.The single chain antibody(sc Fv)which targets HER-2 retains the partial affinity of natural antibody and has the characteristics of small molecular weight and easy to reach the tumor,so it is a good choice for targeting carrier of immunotoxin.Melittin is the main component of bee venom.Ithas small molecular weight,low immunogenicity and activity of rupturing membrane and inducing tumor apoptosis,So Melittin is the first choice for toxin molecules because of its good cytotoxic effect for the tumor cells.However,the hemolytic activity of Melittin has limited its application in clinical treatment.In our laboratory,we acquire the mutation of Melittin(Mmut)that have the ability of killing and no hemolyti activity by optimizing the molecular structure of Melittin.In this article,the Mmut is used as an effective molecule.In this study,the sc Fv which regard HER-2 as the target was selected as the targeting vector,and the mutation of Melittin(Mmut)was selected as the effective molecule,they were combined to immunotoxin(sc Fv-Mmut).We obtained target protein by inducing Pichia pastoris to express,and preliminary studied the antibreast cancer activity of sc Fv-Mmut through the in vitro experiments.The specific experiments include the following aspects:(1)The expression system of Pichia pastoris was established for sc Fv-MmutFirst,the Pichia pastoris expression plasmid p PIC9K/sc Fv-Mmut was constructed,then plasmid was linear by restriction enzymes Sac ?,last it's transformed into Pichia pastoris GS115 by electroporation;Positive strains were screened by MM/MD plate and G418 resistance;0.5% methanol was used to induce the expression.The expressed product experienced 15%SDS-PAGE electrophoresis analysis to obtain the engineered strain sc Fv-Mmut1.(2)Purification of immunotoxin sc Fv-MmutFirst,sc Fv-Mmut1 was induced to express by 0.5% methanol,then fermentation supernatant was collected by centrifugation.The target protein solution was obtained at a concentration of 0.42?g·?L-1by ammonium sulfate deposition,affinity chromatography for purification of Ni-Sepharose 6 FF,identification of15%SDS-PAGE and detect of BCA standard protein kit.(3)Activity detection of immunotoxin sc Fv-MmutMTT assay was used to detect the inhibitory effect of immunotoxin sc Fv-Mmut on HER-2 positive breast cancer cell line BT474 and HER-2 negative breast cancercell line MCF-7.The results of MTT assay showed that sc Fv-Mmut had a significant inhibitory effect on the HER-2 positive breast cancer BT474 cell,and was significantly higher than that of HER-2 negative breast cancer MCF-7 cell.We used DNA ladder,DAPI staining and flow cytometer to detect the effect mechanism that immunotoxin sc Fv-Mmut act on BT474.DNA ladder experimental results showed that chromosomal DNA of BT474 cell ruptured,the emergence of DNA ladder meant cell apoptosis;After DAPI staining we observed with fluorescence microscopy,compared the cell nucleus with blank control cells.We found the nucleus of BT474 which contact with sc Fv-Mmut is shrinking and form a lot particulate matter,indicating cell apoptosis;the results of flow cytometry cycle detection showed that BT474 cell stay in the S phase.We used stain to label the nucleus(by Hochest33342),mitochondria(by Mito Tracker? Red CMXRos)and sc Fv-Mmut(by FITC).Distribution of sc Fv-Mmut in cells and ability of targeting cells were observed under confocal laser microscopy.The results showed that sc Fv-Mmut can target tumor cells and go into the cytoplasm.Above all,the recombinant plasmid p PIC9K/sc Fv-Mmut was successfully constructed and the sc Fv-Mmut was successfully expressed in the eukaryotic expression system of Pichia pastoris.The biological activity and mechanism of sc Fv-Mmut show that,the sc Fv-Mmut designed with HER-2 has a good braking effect on tumor,it can specifically target HER-2 positive breast cancer cells and enter the cytoplasm to induce apoptosis.