| The treatment of malignent tumors in the head and neck region is often difficult just because this region has a very special anatomic structure. The special biological feature of tumors in the head and neck region is another factor that causes the difficulty of treatments. The surgical treatment, chemotherapy and radiotherapy are not enough to reduce the mortality rate of some tumors in this region. They may also reduce the life quality of the patients. With the development of the molecular biology and tumor biology, targeted immunotoxin is becoming an important direction in the tumor treatment. The targeted immunotoxin is to use a carrier to transport cytotoxic meterials to tumor cells. The carrier must have special targeted binding ability to the tumor cells. This could make the targeted immunotxin have the ability to kill tumor cells but have no toxic effect to other normal cells. Some materials can be as a carrier of the targeted immunotoxin, such as :monoantibody, hormones. These materials have high binding rate to some tumor cells. The also have simple structre which is very important to construct with toxin gene using gene recombinant technique. The toxin part is classified as chemical toxin, bacterial toxin and radiate toxin according to their characters. The bacterial toxins have strong cytotoxin effect and special structures: they have a binding region, a translationg region and a toxin effect region. It is easy to use carrier instead of the binding region. So the bacerial toxins are often to be used as a toxin part of the recombiant immunotxin. There are two ways for conjunction of the carrier and the toxin parts: one is to use chemical method to conjunct the carrier' protein to the toxin protein. The other is to recombinant the carrier' gene with the toxin gene, then express the recombinant gene in a expression system to express a recombinant protein. The later method may get stable recombinant protein and can be made on a large scale. From the last 80', many targeted recombinant immunotoxin have been made. Some of them have been approved to be used in clinic. The aim of this study is to construct a recombinant toxin using the genes of α-MSH and Ang and test its anti-tumor effects. The surface of malignant melanoma cell has high expression of MSHR which is special receptor of α-MSH.. We use gene recombinant technique to conjunction the gene of α-MSH with the gene of bacterial toxin Ang in PET-20b plasmid. Then conduct the plasmid to E.Coli BL21(DE3)Plyss. Inducing the bacterial with IPTG, the recombinant toxin MSH- Ang was produced. The product was tested by SDS-PAGE and Western blotting analysis. After purification of the products, the recombinant toxin MSH- Ang was tested on melanoma cell B16, laryngeal carcinoma cell Hep-2,stamoch carcinoma cell BCG-823 and Hela cell. Use CHO cell and the expressions of the BL21(DE3)Plyss without plasmid PET-MP as control groups. After 24 hours of culture, observe the cell' morpholity. Results: analysis of digesting with restriction endonucleases and DNA sequencing verified that the junction between two genes had right reading frame. The expression rate of MSH- Ang is 10%. The purification methods have been constructed and the purification rate is above 92%. MSH- Ang have strong toxin to melanoma cell B16, laryngeal carcinoma cell Hep-2, stamoch carcinoma cell BCG-823 and Hela cell, the control groups have no obvious changes . Conclusion: We have successfully constructed a recombinant toxin MSH- Ang which not only have targeted toxin effect to malignant melanoma cell B16, but also have targeted cytotoxin to laryngeal carcinoma cell Hep-2, stamoch carcinoma cell BCG-823 and Hela cell. We guess that these tumor cells have some receptors of MSHR or ACTHR relative, so the MSH- Ang can bind with these receptors. We also construct an purification courses of the product(92%). Our study establish a foundation of anti-tumor biology toxin.
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