ABSTRACT
Based on the computer simulation, we analyzed hydrophobicity, potential epitope of recombined subtypes HIV-1 Env protein (851 amino acids) from Guangxi in China. Compared with conservative peptides of other subtypes in env protein, three sequences (469-511aa, 538-674aa, 700-734aa) were selected to recombine into a chimeric gene that codes three conservative epitope peptides with stronger antigencity, and was constructed in the yeast expression plasmid pPICZB. Chimeric proteins were expressed in Pichia pastoris under the induction of methanol, and were analyzed by SDS-PAGE and Westernblot. The results showed that fusion proteins of three-segment antigen were expressed in Pichia pastoris and that specific protein band at the site of 40kD was target protein, which is interacted with HIV-1 serum. The target proteins were purified by metal Ni-sepharose 4B, and were demonstrated to possess good antigenic specificity from the data of ELISA. This chimeric antigen may be used as research and developed into HIV diagnostic reagents.
Subject(s)
Humans , Blotting, Western , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation, Viral , HIV Seronegativity , Allergy and Immunology , HIV Seropositivity , Allergy and Immunology , HIV-1 , Genetics , Allergy and Immunology , Metabolism , Pichia , Genetics , Recombinant Fusion Proteins , Genetics , Allergy and Immunology , Metabolism , env Gene Products, Human Immunodeficiency Virus , Genetics , Allergy and Immunology , MetabolismABSTRACT
<p><b>OBJECTIVE</b>To study the genome sequence of hepatitis A virus L-A-1 strain which has been applied for live attenuated vaccine production in China, to compare with other HAV strains, to understand some characteristics of L-A-1 strain, and to find the mechanism of attenuation and cell adaptation.</p><p><b>METHODS</b>Genome fragments were prepared by antigen-capture PCR from infected cell (2BS), PCR products were cloned into T vector, sequenced and analyzed by using bioinformatics program.</p><p><b>RESULTS</b>Analysis of the genomic sequences(nt 25-7,418) showed that the open reading frame contains 6,675 nucleotides in length encoding 2,225 amino acids. Sequence homology comparison showed 98.00% and 94.00% homology at nucleotide level, and 98.51% and 98.65% homology at amino acid level with international strains MBB and HM 175, respectively. Through comparison with other attenuated, cell adapted and cytopathic effect (CPE) strains, L-A-1 strain had mutation at nt 152, 591, 646, 687 and insertion at nt 180-181 in 5?NTR and had mutation at nt 3,889 (aa 1 052-Val) in 2B region, these mutations and insertion are molecular basis for cell adaptation; mutation at nt 4,185 (aa 1 152-Lys) in 2C region should be attenuated marker; deletion in 3A region (nt 5,020-5,025) that caused two amino acids deletion is virus fast growth basis.</p><p><b>CONCLUSION</b>Through analyzing L-A-1 strain genomic sequence, certain sites related to cell adaptation and attenuation were found.</p>