Cloning of HBsAg-encoded genes in different vectors and their expression in eukaryotic cells

Abstract

AIM:To compare the efficiency of different plasmids as DNA vectors by cloning three HBsAg-encoded genes into two eukaryotic expression vectors, pRc/CMV and pSG5UTPL/ Flag, and to express HBsAg S, MS, and LS proteins in SP2/ 0 cells, and to establish monoclone SP2/0 cell strains that are capable of expressing S or S2S proteins stably. METHODS:Segments of S, preS2-S, preS1-preS2-S genes of Hepatitis B virus were amplified by routine PCR and preS1-S fragment was amplified by Over-Lap Extension PCR. The amplified segments were cleaved with restricted endonuclease Hind III/Not I followed by ligation with pRc/ CMV, or BamHI/EcoR I followed by ligation with pSG5UTPL/ Flag. After the plasmid vectors were cleaved with the correspond enzymes, the amplified segments were inserted into pRc/CMV or pSG5UTPL/Flag plasmid vectors with T4 DNA ligase. KOZAK sequence was added before the initial ATG code of each fragment using specific primer. The inserted segments in the recombinant plasmids were sequenced after subcloning. BALB/c mice myeloma cells (SP2/0 cell line) were transfected with the recombinant plasmids. The expressions of the different recombinants were Compared by Western-blot, using a monoclonal anti-HBs antibody as the primary antibody and peroxidase-labeled multi-linker as the secondary. Stable SP2/0-pRc/CMV-S or SP2/0-pRc/CMV-MS clones were established through clone screening with G418.

Occult HBV Infection: A Faceless Enemy in Liver Cancer Development

“…The middle (M) lacks the N-terminal 119 aa of L (the pre-S1 sequence), and the small (S) lacks the N-terminal 55 aa of M (the pre-S2 sequence) [ 13 ]. The HBsAg consists of S (S, small surface), MS (medium surface, S+preS2), and LS (large surface, S+preS1+preS2) HBsAg molecules [ 14 ]. The three proteins are post-translationally modified: each of them exhibits a partially glycosylated site in the S domain, an additional glycosylation in the pre-S2 region of the M protein, and the L protein differs from the other envelope proteins by a N-terminal myristylation [ 15 ].…”

Substitution of <i>Pichia pastoris</i>-Derived Recombinant Proteins with Mannose Containing O- and N-Linked Glycans Decreases Specificity of Diagnostic Tests

“…After 4–5 days, the supernatants were harvested by centrifugation. For the expression of enzymatically inactive recombinant Pr3 (rPr3), its active site serine (Ser 176) was changed to an alanine using the overlap extension method [19]. The resulting cDNA was sequenced and subcloned into the P. pastoris expression vector pPicZαA, which was subsequently transformed to the P. pastoris strain KM71, according to the manufacturer’s instructions (Invitrogen).…”

Serodiagnosis of Hepatitis B Virus Using PreS Antigen from Pakistani Isolate SBS001

“…PreS domain has previously been expressed in many hosts, like yeast [11], plants [12]and eukarryotic cells [13], but it is most easy to express this region in bacterial system due to ease of purification and high yield. To simplify the purification method and avoid the degradation of recombinant protein, 6X His tag was added to the carboxyl end of the preS polypeptide.…”