重组人乳铁蛋白基因克隆及表达

目的　构建重组人乳铁蛋白 (HLF)的毕赤酵母分泌表达菌株 ,并对表达产物进行评价。方法　由外周血白细胞总RNA经RT- PCR克隆获得HLF基因结构cDNA ,测序后克隆入酵母表达载体获得质粒pPIC9K- HLF ,线性化后电转化入毕赤酵母 ,经G4 -18 YPD筛选多拷贝后进行甲醇诱导表达。结果　获得基因与GenBank中的人HLF0 1基因基本相符。 2株多拷贝菌株诱导表达产物均能与人HLF抗体反应 ,具有抑制大肠杆菌生长的作用。结论　获得了能分泌有活性HLF的重组人乳铁蛋白 (HLF)的毕赤酵母分泌表达菌株。

Cloning and Expression of Recombinant Human Lactoferrin Gene

Objective To construct a recombinant Pichia pas toris strain for secretory expression of human lactoferrin (HLF) and identify the expressed product.Methods Amplify cDNA from the total RNA of pe ripheral blood leukocyte by RT-PCR,identify by sequencing and clone into expres sion vector pPIC9K.Linearize the constructed recombinant plasmid pPIC9K-HLF and transform to Pichia pastoris by electroporation,then screen positive clones wit h G418-YPD for expression under induction of methanol.Results The sequence of amplified gene was basically consistent with that of HLF01 gene reported in GenBank.Both the expressed products in 2 multi-copy bacterial strai ns reacted with antibody to human HLF and showed significant inhibitory effect o n the growth of E.coli.Conclusion A Pichia pastoris str ain for expression of recombinant HLF protein was obtained.