Detection, Cloning And Expression Of α-melanocyte Stimulating Hormone Receptor Of Human Melanoma Cell

The targeting toxin is a new type of anti-tumor chemotherapy reagent. It is consist of a targeting polypeptide carrier covalently linked to a peptide toxin. It can kill malignant cells specifically. The most important aspect of targeting toxin is it's specificity, that means the toxin can kill the malignant cells, at the same time it doesn't hurt normal cells, so the receptors on the malignant cells which can bind the carrier of targeting toxin take important role to help the targeting toxin kill the tumors. To find these receptors and make clear the function of them are the most important work before develop a new targeting toxin. The motive of this experiment is to detect the expression of alpha melanocyte stimulating hormone receptor(MC1R)on 5 kinds of human tumor cells : HeLa,SK-BR-3,A549,HepG2,A375. That is to find the special target MSH can bind. Cloning the MC1R gene and study the expression of it in E.coli and insect cell. The results of the above mentioned experiments will provide a basic conditions for future research of MC1R and the development of targeting toxin. Experiment 1: The protein of MC1R was detected on 5 kinds membrane of human tumor cell: HeLa,SK-BR-3,A549,HepG2,A375 by using of 125I labeled α-MSH radioligand binding assay. Design a pair of primers according to the MC1R gene sequence published in Genebank and amplify the full length MC1R gene from the 5 tumor cells by RT-PCR . Cloning the amplified MC1R gene into pMD-18T vector and identify by PCR and digestion with restriction enzyme. Select the positive clones with MC1R gene inserted for nucleotide sequencing. Compare the sequence with the MC1R gene sequence published in Genebank. The results showed that one kind of receptor on melanoma cell line A375 can bind 125 IαMSH. The other cells'MC1R were not find out. The DNA sequence cloned was 98% consistent with the MC1R gene sequence published in Genebank[Arg67Gln ( g/a ),Asp84Glu(c/a) ,Val 92 Met(g/a) ,Arg151Cys(c/t),Arg163Gln (g/a),Thr314Thr(a/g)]. The expression of MC1R protein and MC1R mRNA are identified only in human melanoma cell line A375 among the five kinds of human tumor cells. Experiment 2: To clone the full length cDNA of alpha melanocyte stimulating hormone recepto(rMC1R)by PCR. Digest the amplified cDNA with restriction enzyme and insert it into Pet-20b for expression in BL21(DE3) Ecoli. Design a pair of primers according to the MC1R gene sequence published in Genbank(EMBL / GenBank acc.no.X67594)and amplify the full length MC1R gene from pMD-18T-MC1R by PCR anddigestion with restriction enzyme EcoRI and NcoI, clone the gene into plasmid Pet-20b, transform it into the vector BL21(DE3) for expression under induction of IPTG. Identify by SDS-PAGE and 125I labeled α-MSH radioligand binding assay. The Results showed that a significant band with a relative molecular weight of 35kDa was observed. The radioligand binding assay proved that expressed protein cann't bind 125 IαMSH. The MC1R gene has been expressed in E.coli but it doesn't have the biology function. Experiment 3: To clone the gene of alpha melanocyte stimulating hormone receptor(MC1R)into pFASTBAC1 for expression in Sf9 insect cells. Design a pair of primers according to the MC1R gene sequence published in Genbank(EMBL / GenBank acc.no.X67594) and amplify the full length MC1R gene from pMD-18T-MC1R by PCR and digestion with restriction enzyme EcoRI and XbaI, clone the gene into plasmid pFASTBAC1, transpose it into a shuttle vector DH10BAC. Transfect Sf9 cells with the recombinant plasmid Bacmid-MC1R. Identify by SDS-PAGE and 125I labeled α-MSH radioligand binding assay. The Results showed that the MC1R protein expressed was collected by centrifugation and detected by SDS-PAGE. A significant band with a relative molecular weight of 35kDa was observed. The radioligand binding assay proved that expressed protein can bind 125 IαMSH. The MC1R gene has been successfully expressed in insect cells. To sum up, The MC1R was identified only in human melanoma cell line A375 among the five kinds of human tumor cells. The radioligand binding assay proved that MC1R can bind 125 IαMSH, αMSH has a good...