Expression, Identification And Purification Of P21ras Proteins And Production Of Their Polyclonal Antibodies

The gene Ras is an important oncogene and the name is derived from ratsarcoma. The human ras gene family consists of three identified members,H-ras,K-ras and N-ras,which locates on the twelfth,eleventh and first chromatosome respectively. Human H-ras and k-ras are the homologues of that H-ras and K-ras sequences originally isolated from Harvey and Kirsten strains of rat sarcoma viruses,lt is well known that they encode the proteins of 188 to 189 amino acids, approximately 21 kilodaltons(P21ras),and the sequences are homologous by 85%.Three proteins of P21ras are signal transmissing proteins,regulating the differentiation and proliferation of cells,and the function is transient under normal conditions.Nevertheless,they can continuously induce the normal cells to differentiate,proliferate and malignantly transform,when Ras mutates or over-expresses.It also has been demonstrated that Ras usually over-expresses in many oncomas,which suggests that over-expression of ras is highly correlative with tumors' genesis and development.Therefore,we make a hypothesis,that the intracellular anti- body of p21ras can selectively counteracts with p21ras,which blocks up the signal transduction and inhibits proliferation of tumor cells,In this study,we have constructed the prokaryotic expressing plasmid of p21ras proteins and producted polyclonal antibodies.The three genes of ras were obtained by RT-PCR and gene synthesis,each with the enzymes BamHI and Hindâ…¢.After digested with BamHI and Hindâ…¢,the H,K and N-ras containing fragments sites were ligated respectively into the vector PET-28a(+)though T4 ligase,which also has been digested with the same restriction enzymes.Restriction enzyme analysis and sequence analysis showed the recombinant plasmids containing ras genes which were the same as we designed previous.Then recombinant plasmids were transformed into the bacterium BL21(DE3)and screened by kanamycin,induced and expressed by IPTG.SDS-PAGE analysis showed three proteins could express effectively in Escherichia coli and the apparent molecular weight was 23KD.Western blot manifested expression products of three genes could be distinctively responsible to anti-p21ras antibodies.The inclusion body of p21ras might be purificated by NI²⁺-NTA resin with the mild denaturant Urea,the purity of which is more than 95%.New Zealand albino rabbits were immuned with purified p21ras protein respectively.Before being immuned,the rabbits' serum was extracted as negative control.We continued to immune rabbits during the first,second,fourth,sixth week.It was lucky to find the antiboby producted at the second week,the highest level at the sixth week.The immunological activities of the three proteins were confirmed by Western blot.In this study,we have succeeded in constructing prokaryotic expressing plasmids of p21ras proteins and producting the polyclonal antibodies and testing their immunological activities.And this provides a scientific base for studying p21ras further in our next research.