Prokaryotic Expression And Polyclonal Antibody Preparation Of BC047440 And Study On Its Expression In Human Hepatocellular Carcinoma

It is known that human hepatocellular carcinoma is one of the most common malignant tumors at our Country, and the present treatment stategy is not ideal. Although great effort has been made, the pathogenesis of hepatocellular is still largely unknown BC047440 highly expressed in hepatocellular carcinoma ,, is a novel gene previously identified by us through SSH. We found that the expression of BC047440 in hepatocellular carcinoma was higher than that in their adjacention cancerous tissue,and this gene is also relevant to the proliferation of hepatocellular carcinoma.To study the effectiveness of BC047440 in genesis and development of hepatocellular carcinoma,we propose to cloning,Prokaryotic expressing and prepareing polyclonal antibody of BC047440,then detecting the expression of BC047440 using western blotting and immunohistochemical technique.We also propose to analyses the relationship between BC047440 expression and patho-clinical character, approach the effectiveness of BC047440 in carcinogenesis and development of hepatocellular carcinoma.Objective: The aim of this study was to clone a human BC047440 gene and to obtain fusion protein of BC047440, through the expression of this gene,then to get the rabbit-anti-human BC047440 polyclonal antibody from the immune rabbit with this fusion protein. Furthermore, we detected the serum BC047440 with this polyclonal antibody in clinical patients.Methods:1,we anabiosis and cultivante the human HepG2 cells,Then we extracted the total RNA from the human HepG2 cells. Using theRT-PCR, we got the fragment of human BC047440 gene with thecoding region, then the target gene was cloned into an Prokaryotic expression vector pET-28a(+) by using the recombinant DNA technique and it was confirmed by DNA sequence analysis and by restriction enzyme digestion. After induced by IPTG, the fusion protein was expressed in E.coli. The fusion protein was purified by affinity chromatography using amylose resin and testified its antigencity by Western-blot.2,we immunized rabbit with purified fusion protein emulsified with an equal volume of Freund's immunoadjuvant. After the rabbit was purified fusion protein as immunogen for 4 times injected with the we collected the immune sera of rabbits. After we separated the sera from the blood and the antibody (IgG) from the immune sera were purified by ammonium sulfate fractionation,then the parified antibodies of the rabbit-anti-human BC047440 polyclonal antibodlies were obtained with the purified antibodies, we used the indirect sandwich enzyme immunoussay to detecte the serum BC047440, then detect the purity and activity of BC047440.3,we detected the expression of BC047440 polyclonal antibody in 58 cases of clinical patients using western blotting and immunohistochemical technique. Further more,analyses the relationship between BC047440 expression and patho-clinical character, approach the effectiveness of BC047440 in carcinogenesis and development of hepatocellular carcinoma.Results: The results of our experiments were as follows:1. We obtained the gene fragment of BC047440, which containes the coding region, and was identified by DNA sequence analysis.2. We constructed the clone of the recombinant and its expression of fusion protein, with antigenicity was confirmed by using the Western-blot analysis.3. We obtained the purified fusion protein, by using the affinity chromatography method and it was identified by SDS-PAGE analysis. Then we used the purified fusion protein as immunogen to immunize the rabbits.The purified specific rabbit-anti-human BC047440 polyclonal antibodies were obtained from the immune sera of rabbits.4. Using recombinant protein as antigen when detected by ELISA, the results showed the titer of antiserum against BC047440 was about 1:256 000.5. Agar immunodiffusion test showed a single precipitation band was formed between antiserum against BC047440 and immunogen.6. Western blotting indicated that the antiserum against BC047440 could bind with the expressed recombinant protein specifically. The result showed the prepared polyclonal antibodies had better immunoreactivity.7. Cellular expression of BC047440 was determined using immunohistochemistry technique on paraffin-embedded tissue sections from hepatocellular carcinoma tissues and their adjacent non-cancerous tissues. The staining for BC047440 was all found to be cytoplasmic in the hepatocellular carcinoma and adjacent non-cancerous tissues. And expressed higher in hepatocellular carcinoma tissues than adjacent non-cancerous ones.8. Expression of BC047440 was determined using immunohistochemistry technique in 58 patients,47cases of positive expression in hepatocellular carcinoma were obtained and 12 cases of positive in adjacent non-cancerous tissues.otherwise,in normal liver tissues,the cases of positive were 0.9. Using immunohistochemistry detection,there was close relationship between BC047440 protein expression and clinicopathologic findings of liver cancer,including pathological class,envelope invasion or portal vein invasion(P<0.05). And there was non-close relationship between BC047440 protein expression and size of tumor,AFP or HBsAg quantitation.(P>0.05).10. Using immunohistochemistry detection, patients of hepatocellular carcinoma obtained lower survival rate with positive expression of BC047440(P<0.05).Furthermore, Patients with both positive expression of BC047440 and portal vein invasion obtained much lower survival rate than the ones with both not(P<0.05). Patients with both positive expression of BC047440 and envelope invasion obtained much lower survival rate than the ones with both not(P<0.05).11. Expression of BC047440 was determined using western blot in 58 patients, Quantity of BC047440 expressiom was much higher in hepatocellular carcinoma tissues than that in adjacent non-cancerous tissues or normal liver tissues(P<0.05 ).12. Using western blotting detection,there was close relationship between BC047440 protein expression and clinicopathologic findings of liver cancer,including pathological class,envelope invasion or portal vein invasion(P<0.05). Conclusion: The fusion protein BC047440,that was expressed by the recombinant has immunogenicity. The BC047440 polyclonal antibody was obtained from the immune sera of rabbits. BC047440 may be related with the genesis and development of human hepatocellular carcinoma probably by overexpression. The proteinic expression of BC047440 may be related to the growth, infiltration and metastasis of human hepatocellular carcinoma.