大肠杆菌天门冬酰胺酶Ⅱ的基因克隆与表达

Molecular Cloning and Expression of ansB,the Escherichia coli GeneEncoding L-Asparaginase Ⅱ

本文报道用一对与大肠杆菌编码Ｌ－天门冬酰胺酶Ⅱ（Ｌ－ＡｓＰｓⅡ）的基因ａｎｓＢ两侧序列互补的寡核苷酸Ｌ１、Ｌ２作引物，以Ｌ－ＡＳＰｓⅡ的野生型生产菌ｃＰＵ２１０００９染色体为模板，经ＰＣＲ扩增得到长约１．６Ｋｂ的ＤＮＡ片段。该片段通过以ＬＰ１、ＬＰ２为内引物的ＰＣＫ反应，证明包含了ａｎｓＢ基因的编码序列。将此片段经ｘｂａ１、ＨｉｎｄⅡ消化，插入质粒ＰＵＣ１８、ＰＵＣ１９上构建重组质粒ＰＵＡ１８、ＰＵＡ１９，分别转化Ａｓ１．１１８７，Ａｓ１．１１９３，ＨＢ１０１，９６３７，ＪＭ１０７，７１／１８，ＣＰＵ２１０００９等宿主菌，筛选得到Ｌ－ＡＳＰｓⅡ酶活力明显提高的８株阳性转化子。在１Ｌ生物反应器中，ＬＢ培养基３７℃下培养２４ｈ，其酶活在６．２～３１．８１Ｕ。ｍｌ之间。其中ＣＴＡ８表达Ｌ－ＡＳＰｓⅡ水平最高，达到３１．８１Ｕ／ｍｌ．各基因工程菌株较之野生型生产菌体ＣＰＵ２１０００９，其生产Ｌ－ＡＳＰｓⅡ水平提高了５～２６倍。而且ＩＰＴＧ诱导对工程菌表达Ｌ－ＡＳＰｓⅡ的水平几无影响。ＳＤＳ－ＰＡＧＥ测得工程菌表达的Ｌ－ＡＳＰｓⅡ分子量约为１４０Ｋｄ．薄层扫描结果显示：ＣＴＡ７、ＣＴＢ８、ＣＴＡ９所产生的Ｌ－ＡＳＰｓ?

y PCR technique,1.6 kb DNA fragment was polymerized with wild-type E,coli CPU 210009 chro-mosome DNA as template and with synthetic oligon ucleotide Li、 L2 as primer. The Agarose Gel Elec-trophoresis and PCR result with LP1 , LP2 as primer proved that this DNA fragment contains ansB. Thenthe DNA fragment was digested by restrictive endonuclease Xba 1/ HindⅢ , and inserted into PUC 18、PUC 19 poly-linker site, The chimeric plasmid PUA18、PUA 19 were constructed. The 7 species of genet-ically-engineered strains were obstrained by PUA 18、PUA 19 transforming sevreal hosts as As1.11 87,As1.1193, HB101, 9637 , JM107, 71/18 CPU210009, Their L-ASPs Ⅱ enzyme activities were be-tween 6.2～31.8 IU/ml. The result showed that the L-ASPsⅡ enzyme activities of recombinantshave increased as high as 5～26 times compared those of with wild-type strain CPU210009. The molec-ular weight of the purified L-ASPs Ⅱof recombinants were estimated to be about 140kd by the resultsof SDS-PAGE, The proportions of the L-ASPs Ⅱof CTA7、CTA8 、 CTA9 were proved to be 28.3%,35.7%, 33,6%in the total lysated protein of vectors by thin-layer screening.