Preparation Of Recombinant Human Angiogenin Protein And Polycional Antibody And Primary Study In Tumor Detection

Angiogenin is one of the angiogenic factors that have been shown to stimulate blood vessel formation, and is the only one thus far to be identified and purified based solely on its angiogenic activity and its ability to induce angiogenesis is strong. Angiogenin belongs to a protein superfamily of RNase and it is the only angiogenic factor known to exhibit nbonucleolytic activity. A large numbers of studies show that angiogenin is very necessary for many different cancers occurrence, and is related closely to the rapid development, metastasis, progression and prognoses of cancers. What's more, angiogenin always overexpressed in the serum of many tumors patients. Thus in the studies of tumor, serum angiogenin expression level become a kind of specific marker of tumor, it has important significance to the early diagnosis of tumor and the check-up of tumors on a large scale.In this study, the human angiogenin cDNA was amplified by RT-PCR method from cultured human malignant melanoma cells A375 and inserted into the expression vector pET28- α (+) after it was proved to be correct by DNA sequencing . Then, the constructed gene was transformed into E.coli BL21 host bacteria. After having been induced by IPTG, the recombinant protein fused with His6 at its N-terminal was expressed successfully. Owing to possessing 6-His tags, the expressed protein can be purified by Ni²⁺ resin according to the high affinity of His6 with Ni²⁺. From the analysis of the antigenicity through Western blot we found the purified recombined protein could bind angiogenin - antibodies specifically. The purified recombined protein immunized habits and polycional antibody was produced.We use the purified recombined protein and polycional antibody to detect the angiogenin expression levels in the serum of the tumor patients by the competitive ELISA. The results of the detection indicated it had good effect in diagnosing liver cancer, gastric cancer and leukaemia, and it could be a rapid, effective and cheap method for tumor diagnosis.