富集液中六六六(γ-BHC)脱氯基因的克隆、表达及降解试验

Cloning, expression of the gene of dehydrochlorination of BHC from enrichment and its degradation experiment

从长期受六六六污染的土壤中获得高效降解六六六的富集液 ,其对六六六 4种异构体的降解效果均为10 0 % ,但至今未能获得纯培养。根据国外报道的六六六脱氯基因linA序列 ,设计并合成一对引物 ,通过PCR技术从富集液总DNA中扩增了 4 71bp的基因片段 ,命名为linN ,测序结果表明linN与报道的脱氯基因linA和linA2的同源性达 99% ,与linA1的同源性达 97%。将linN定向克隆到pET 2 9α表达载体中 ,转化至E .coliBL2 1,经IPTG诱导后可表达分子量约 17kD的蛋白 ,表达产物占菌体总蛋白的 30 %左右 ;诱导后转化子的降解能力明显提高 ,粗酶也有很好的降解效果 。

A high effective enrichment that could degrade four isomers of BHC completely was got from the soil polluted by γ-BHC, but the pure culture was not obtained yet. The 471bp sequence of linN was amplified from the total DNA of enrichment by polymerase chain reaction(PCR). The nucleotide sequence analysis showed that the linN gene had high homology with reported linA up to 99%. Then the amplified fragment linN was cloned in the proper orientation into the site between NdeⅠ and HindⅢ of pET-29α via restriction endonuclease NdeⅠ and HindⅢ. The recombinant was transformed into its host E.coli strain BL21 and a recombinant protein of about 17kD was highly expressed and showed high ability of degrading γ-BHC after induced by IPTG. The expressed protein occupied about 30% of the total bacterial protein. The cell extracts also showed some ability of degradation of γ-BHC. It offered basic theory for the isolation of pure culture and the construction of genetic engineering microorganisms.