Expression, Purification And Characterization Of Recombinant Immunotoxin MSH-PE38KDEL Which Specifically Targets Melanoma

Malignant melanoma is one of the malignant tumors with the fastest incidencegrowth. So far, the most effective therapy to the metastatic melanoma has not beendiscovered. Recombinant toxin is expected to become one of the most effective(assisting) therapy to melanoma for its high particularity and strong cytotoxity,especially to the metastatic melanoma. This study concentrated on the specificcombination of α-MSH with MC1R on the surface of malignant melanoma, as well asthe dozens of times’ gap between the binding site of MC1R on the surface ofmalignant melanoma and that of normal cells and other tumors, to make it as aspecific targeting carrier of malignant melanoma. The study also used pseudomonasexotoxin (PE) as the toxic part of immunotoxin, to build a recombinant toxin called asMSH-PE38KDEL, expressing melanoma targeting toxin in the way of geneticengineering.Construction and expression of recombinant toxin—MSH-PE38KDEL. In this study,the linker of MSH and PE40was optimized by bioinformatics analysis, to ensure thattheir relative structure and function were not to be affected. Then PE40was modified,to reduce its immunogenicity (PE38) and increase its activity (PE38KDEL). Next thegene (MSH-PE38KDEL) was constructed and linked to the expression vectors—pET28a and pHis1525, to construct two recombinant plasmids—pET28a(+)-MSH-PE38KDEL and pHis1525-MSH-PE38KDEL, and they were expressed in theexpression host—Rosetta-gamiTM2(DE3) and WH320. By SDS-PAGE analysis andactivity determination, the recombinant plasmid with high expression andtoxicity—pET28a(+)-MSH-PE38KDEL was finally screened out. The plasmid wastranslated into Rosetta-gamiTM2(DE3) cell after it was determined with the correctsequence, and IPTG was used to induce fusion protein MSH-PE38KDEL expression,with SDS-PAGE as the determination on the expression products. Meanwhile, thesupporting software of gel image analysis system was applied to the analysis on expression level of MSH-PE38KDEL fusion protein, which took10%of the wholebacteriaPurification of recombinant toxin MSH-PE38KDEL. The Western Blot was usedto make somatic analyze after ultrasonic disruption to prove the soluble expression offusion protein. Purification methods such as somatic ultrasonic disruption,centrifugation, hydrophobic chromatography, ion exchange, molecular sievechromatography were adopted on the protein after expression. The purity quotient ofMSH-PE38KDEL with SDS-PAGE obtained over90%.The in vitro antimelanoma activity of recombinant toxin MSH-PE38KDEL.The study tested the in vitro biological activity of human melanoma A875cells andmurine melanoma B16cells with high expression of α-MSH receptor, comparingthem with CNE, MCF, and2BS cells, whose α-MSH receptor expressions are low, inorder to prove its targeted killing effect. MTT test results showed that the killingeffect of MSH-PE38KDEL on melanoma cells A875and B16was over85%, and0onthe human normal2BS cells.In vivo antimelanoma activity of recombinant toxin MSH-PE38KDEL. Themurine melanoma B16-cell was adopted in nude mice to establish a melanoma highlymetastatic animal model. The tumor-bearing mice were administrated with the routesof peri-tumor injection and intravenous injection. The result was that after10consecutive days of daily injection with the MSH-PE38KDEL concentration of1.1mg/mouse, the tumor growth was inhibited significantly with the rate of over90%.Meanwhile, the rate of disappearance in the group of peri-tumor injection was30%,and in the group of intravenous injection—that was40%. The HE staining resultshowed that MSH-PE38KDEL effectively inhibited the metastasis of melanoma B16cells in livers. The ultra structure of liver electron microscopy revealed thatMSH-PE38KDEL inhibited the damaging effects of melanoma B16cells on the livercells. Recombinant toxin MSH-PE38KDEL owned obvious antimelanoma effects invivo, with low toxic and side effects. The pathology results and electron microscopyrevealed no damage to the parenchymatous. In all, MSH-PE38KDEL is expected tobe adopted as one of the drug options to the targeting treatment for malignantmelanoma.