title: [Construction of eukaryotic recombinant vector and expression in COS7 cell of LipL32-HlyX fusion gene from Leptospira serovar Lai].
abstract: This study was conducted to construct eukaryotic recombinant vector of LipL32-HlyX fusion gene from Leptospira serovar Lai and express it in mammalian cell. Both of LipL32 gene and HlyX gene were amplified from Leptospira strain O17 genomic DNA by PCR. Then with the two genes as template, LipL32-HlyX fusion gene was obtained by SOE PCR (gene splicing by overlap extension PCR). The fusion gene was then cloned into pcDNA3.1 by restriction nuclease digestion. Having been transformed into E. coli DH5alpha, the recombiant plasmid was identified by restriction nuclease digestion, PCR analysis and sequencing. The recombinant plasmid was then transfected into COS7 cell whose expression was detected by RT-PCR and Western blotting analysis. RT-PCR amplified a fragment about 2000 bp and Western blotting analysis found a specific band about 75 KD which was consistent with the expected fusion protein size. In conclusion, the successful construction of eukaryotic recombinant vector containing LipL32-HlyX fusion gene and the effective expression in mammalian have laid a foundation for the application of Leptospira DNA vaccine.
authors.affiliation: Research Unit of Infection and Immunity, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
authors.forename: Bi
authors.initials: B
authors.lastname: Huang
authors.forename: Lang
authors.initials: L
authors.lastname: Bao
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhong
authors.forename: Huidong
authors.initials: H
authors.lastname: Zhang
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D050939:Gene Fusion; D005822:Genetic Vectors; D018257:Helix-Loop-Helix Motifs / Q000235:genetics*; D007919:Leptospira / Q000235:genetics*; D008074:Lipoproteins / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; C406589:LipL32 protein, Leptospira; D008074:Lipoproteins; D011993:Recombinant Fusion Proteins