Objective: To study the effect of sequence variability between different genotypes of HCV on the antigenic properties of the NS5a protein of strong antigenic re- gion at position 2212-2313 by using recombinant protein...Objective: To study the effect of sequence variability between different genotypes of HCV on the antigenic properties of the NS5a protein of strong antigenic re- gion at position 2212-2313 by using recombinant proteins. Methods: Thirteen representative sequences from HCV genotypes 1 to 6 were selected to design synthe- tic genes encoding for this antigenic region, These genes were assembled by PCR from synthetic olionu- cleotides and expressed in E. coli as hybrid proteins with glutathione S-transferase. All 13 fusion proteins were purified from bacterial lysates and used to test a panel of anti-HCV positive sera (n=61) obtained from patients infected with HCV genotypes 1 through 6. Results: A comparison of sequences derived from dif- ferent HCV genotypes showed that the primary struc- ture of this strong antigenic region is highly variable. Percent homology between different genotype se- quences varied from 40. 4% to 72. 5%. All but one protein immunoreacted with 62% to 93% of serum samples. Although a variable degree of genotype spe- cific antigenic reactivity was detected, only one pro- tein demonstrated a noticeable preference to immu- noreact with antibodies against the homologous HCV genotype. On the other hand, closely related proteins derived from the same subtype or genotype immuno- reacted with significantly different efficiency with HCV antibodies. Conclusions: Different genotype HCV genes were successfully cloned, expressed and purified. Se- quence variability has a profound effect on the anti- genic properties of the HCV NS5a immunodominant region. This finding should be considered in the de- velopment of diagnostic tests for the efficient detec- tion of anti-HCV activity in serum specimens.展开更多
文摘Objective: To study the effect of sequence variability between different genotypes of HCV on the antigenic properties of the NS5a protein of strong antigenic re- gion at position 2212-2313 by using recombinant proteins. Methods: Thirteen representative sequences from HCV genotypes 1 to 6 were selected to design synthe- tic genes encoding for this antigenic region, These genes were assembled by PCR from synthetic olionu- cleotides and expressed in E. coli as hybrid proteins with glutathione S-transferase. All 13 fusion proteins were purified from bacterial lysates and used to test a panel of anti-HCV positive sera (n=61) obtained from patients infected with HCV genotypes 1 through 6. Results: A comparison of sequences derived from dif- ferent HCV genotypes showed that the primary struc- ture of this strong antigenic region is highly variable. Percent homology between different genotype se- quences varied from 40. 4% to 72. 5%. All but one protein immunoreacted with 62% to 93% of serum samples. Although a variable degree of genotype spe- cific antigenic reactivity was detected, only one pro- tein demonstrated a noticeable preference to immu- noreact with antibodies against the homologous HCV genotype. On the other hand, closely related proteins derived from the same subtype or genotype immuno- reacted with significantly different efficiency with HCV antibodies. Conclusions: Different genotype HCV genes were successfully cloned, expressed and purified. Se- quence variability has a profound effect on the anti- genic properties of the HCV NS5a immunodominant region. This finding should be considered in the de- velopment of diagnostic tests for the efficient detec- tion of anti-HCV activity in serum specimens.
