钩端螺旋体LipL21基因真核表达载体pcDNA3.1(+)/LipL21的构建与鉴定

目的构建钧端螺旋体外膜脂蛋白LipL21基因片段的真核表达载体，寻找新的预防钧端螺旋体感染的候选疫苗。方法应用PCR技术从钧端螺旋体黄疸出血群赖型56601株基因组模板中扩增LipL21基因，纯化回收后克隆入pUCm-T载体，再亚克隆入真核表达载体pcDNA3．1（＋）。结果双酶切及测序鉴定证明成功构建了LipL21真核表达载体pcDNA3．1（＋）／LipL21，DNA测序显示重组质粒含有561bp的目的基因片段，读码框架正确，无碱基错配及移码突变。测序后结果经与GenBank登录的序列做blast比较，载体上所连目的基因片段序列与GenBank登录的LipL21基因序列完全一致。结论成功构建了钧端螺旋体LipL21基因真核表达载体pcDNA3．1（＋）／LipL21。

Identification and Construction of Eukaryotic Expression Vector pcDNA3.1(+)/LipL21 Containing LipL21 Gene of Leptosipra

Objective To construct the eukaryoctic expression plasmid pcDNA3.1( )/LipL21 containing the outer membrane Lipoprotein LipL21 of the Leptospira interrogans serovar Lai,and provide a new candidate antigen for preventing leptospirosis.Methods LipL21 Gene was amplified from the genomic DNA of Leptospira interrogans serovar Lai,strain Lai 56 601 by polymerase chain reaction(PCR),and the gene was inserted into cloning vector pUCm-T.The inserted LipL21 gene was subcloned into appropriate site of pcDNA3.1( ) vector.The positive clones were screened after being identifid with restrictive enzymes and sequence analysis.After being sequenced with DNA auto-sequence analysis instrument,the amplified DNA sequence of LipL21 was searched for alignment with NCBI Blast program.Results The target gene LipL21 segment about 561bp was obtained successfully.Homology of the nucleotide and putative amino acid sequence of LipL21 gene between Leptospira interrogans serovar Lai,strain Lai 56601was 100%.Conclusion The recombinant plasmid of pcDNA3.1( )/LipL21 was constructed successfully.