Study On Variation And Clinic Of Functional Gene Of HBV Of The Diverse-Race Patients With Chronic Hepatitis B From Ethnic Minority Areas In Yunnan

Objective This paper is intended to investigate, by employing molecular biology technique, into variation and clinic of functional gene of HBV preS2/S and C gene of the 34 diverse-race patients with chronic hepatitis B (CHB) from ethnic minority areas in Yunnan Province, P. R. China. The study is to explain HB's pathogenesis of chronicity and deterioration, and provide a theoretical basis for designing safe and effective vaccines for human hepatitis B, which is of great significance in improving the health level of the ethnic people of China.Methods The fragments of HBV preS2/S and C genes were inserted into vector pBS (pBluescript II SK, pBS) after being amplified by PCR (polymerase chain reaction, PCR) from HBV DNA , which were obtained from serum-samples of 34 CHB patients (17 diverse-race patients and 17 han patients ) from ethnic minority areas in Yunnan, and the cloned preS2/S and C genes were sequenced and the functional significance of HBV variants were studied. Then the preS2/S and C genes were cloned and transformed into E.coli TOP 10 to express .The corresponding proteins were identified through the methods of SDS-PAGE and Western blot.Results The sequences of HBV preS2/S and C genes from 34 cases were 846(with 49.1% of GC) and 552 (with 46.1% of GC nucleotides (nt) in length, and encoded 281 and 183 amino acids (aa) respectively. Compared with the HBV subtype sequence ( adr, adw, ayw and ayr) from GenBank , the HBV preS2/S and C genes were homologous to aywl in sequence up to 97.5%~98.6%, and 94.5%~97.8%, respectively. The HBV preS2/S and C genes in 17 diverse-race patients with CHB were not significantly different from those in 17 Han patients with CHB from ethnic minority areas (p>0.05). In all 34 cases, the 122th and 160th amino acid residues of HBV S region were found to be Arginine(AGA) at nt 518-520 and Lysine(AAA) at nt 632-634, respectively. The nucleotide variation of HBV C gene from 34 cases caused amino acid substitutionsamong codons 27-63, 80-110 , and 135-153 in T- and B-cell epitopes. In 29 CHB patients, HBV Core region identified being the amino acid substitutions of I to V , Y to N ,G to V, P to Q and T to A at positions 27,38,63,135 and 147 in T-cell epitope which change the structure of the HBV core due to A to G ,T to A, G to T, C to A ,A to G transitions at nucleotide positions 1979 , 2012 ,2088 ,2304 and 2339, respectively. Whereas as to the other 5 chronic severe hepatitis B , there were S to G and I to L at amino acid positions 87 and 97 in B-cell epitope which change the structure of HBV core due to A to G and A to C transitions at nucleotide positions 2159 and 2189. The genes we have obtained, namely preS2/S and C genes, were cloned into p A PR-S2S and p A PR-C plasmid and were expressed in E.coli TOP 10. The high level bacterial expression products for two proteins encoded preS2/S and C genes with relative molecular weights of 31000 and 21000 were achieved by SDS-PAGE. The target proteins in the research were verified to have preS2/S and Core epitopes by Western blot.Conclusion The HBV DNA of the diverse-race patients with CHB from ethnic minority areas in Yunnan, China was found to be subtype aywl ( geneotype B). Sequence comparisons showed that the HBV DNA in diverse-race patients with CHB were not significantly different from those in Han patients with CHB from ethnic minority areas (p>0.05) , which means that HBV was not related to any of the diverse-races, and that no HBV difference was found among patients from different races. The HBV Core variations in T- and B-cell epitopes showed clustering, which were associated with continuous liver damage and deterioration. The HBV functional genetic variation may be related to the pathogenesis, to the alteration of immunity response and to drug-resistance. The target proteins encoded preS2/S and C genes in the research proved to have antigenicity by Western blot. Findings of the study have for the first time revealed the S'antigen amino acid (aa) 121-147 with CRTCTTPAQGTSMFPSCCCTKPMDGNC epitopes, the C'antigen aa 75-83 wit...