曼氏血吸虫卵可溶性抗原基因克隆的研究

GENE CLONING OF A SOLUBLE EGG ANTIGEN OF SCHISTOSOMA MANSONI

本文报导以曼氏血吸虫(Schistosoma mansoni)成虫mRNA为模板,通过逆转录酶促法合成cDNA。将虫源cDNA插入载体λgtll噬菌体基团组中编码β-半乳糖苷酶的LacZ基因区形成重组DNA,然后导入受体细胞E.coli KM392pmc9(lon^+)建立基因库,并从中筛选出若干株血吸虫抗原基因克隆。一株被兔抗虫卵可溶性抗原高免血清检获的基因克隆,其虫源基因片段经分子杂交证明系单拷贝基因。重组基因在溶原菌株中所表达的融合蛋白通过免疫印迹转移试验(EITB)证明与相关抗体具有很强的结合能力。DNA序列分析揭示该基因编码的多肽系由239个残基组成,其分子量理论推导值为28.7KD,化学性质呈强硷性和强亲水性。引人注意的是此多肽涟中含有一个由7个“精氨酸—甘氨酸”重复组成的结构独特片段。

A cDNA library, constructed from raRNA isolated from adult Schistosoma mansoni was screened with a variety of antisera. Positive clones were recognized by either chronic human infection serum(IHS), chronic mouse infection serum ( IMS ) , rabbit anti-soluble egg antigen serum ( RαSEA ) , rabbit anti-S. japonicnm serum ( RαSj ) , rabbit anti-uninfected Biomphalaria glabrata serum ( RαBg ) or Bovine anti-Fasciola hepatica serum ( BαFh ) . One recombinant clone E-b was studied in detail. The foreign schisto gene of this clone was proved to be a single copy gene which could be hybridized with fragments from the adult worm genomic DNA. A fusion protein of 145 KD was expressed successfully in the lysogenized host cells, E. coli MC4100 and proved highly reactive with SEA by enzyme immunotransfer blot ( EITB ) . A Sequence analysis of the gene coding for this schistosome protein was accomplished by dideoxy nucleotide chain termination method and revealed that this gene consisted of 855 base pairs.A deduced amino acid sequence from an open reading frame showed that the polypeptide contained 239 residues in which there was a unique strong basic polypeptide fragment comprising 7 repeats of 'Arg-Gly' forming a quite hydro-philic antigenic determinant.