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PubMed1998

Research on the Diversity of PLA(2) Gene from Agkistrodon halys Pallas.

We used degenerate primers to amplify phospholipase A(2)(PLA(2)) gene by RT-PCR from venom total RNA of Agkistrodon halys Pallas, and screened with B-PLA(2) gene as probe. Finally we isolated three cDNAs containing A-PLA(2) and two other genes showing similar characteristic structure--Asn(49)-PLA(2) and BA-PLA(2). Their complete sequence was determined by bidirectional sequencing and their amino acid sequence was deduced. The amino acid sequence of A-PLA(2) deduced from the cDNA agreed with that determined by protein sequencing except for four residues; Asn(49 )-PLA(2) and B-PLA(2) are very alike (up to 95%), but the Asp(49)-PLA(2) of B-PLA(2) involved in the enzyme reaction is replaced by Asn(49) of Asn(49)-PLA(2). Therefore, it possibly affects the enzyme reaction of Asn(49)-PLA(2). The N-terminus sequence of BA-PLA(2) is highly homologous to B-PLA(2), but its C-terminus sequence is almost the same as A-PLA(2). The successful cloning of these isoenzyme genes not only discloses the structure diversity of PLA(2)(namely DNA modification and recombination), but also provides excellent material for the study of structure-function relationship in PLA(2).

Journal Article↗

PubMed1996

Cloning of the BPLA(2) Gene from Agkistrodon halys Pallas.

First strand cDNA synthesis was primed with synthetic oligonucleotide from total RNA extracted from the gland of the snake Agkistrodon halys Pallas. The pro-BPLA(2) gene was then amplified by PCR and cloned into the pBS-ks vector. Its nucleotide sequence has been determined by analyzing the DNA sequence of three colonies containing the pro-BPLA(2) gene. The deduced amino acid sequence consists of 138 amino acids and agrees with the partly known amino acid sequence except for seven amino acid residues. The successful cloning of the BPLA(2) gene not only has made the determination of its total amino acid sequence possible, but also provided a good basis for further research work in the protein engineering of functional peptides from snakes.

Journal Article↗