title: [Construction and expression of nm23-H1 gene with different enzyme activities and resistant specific shRNA in eukaryotic expression vector].
abstract: OBJECTIVE
  It has been proven that nm23-H1 gene was a tumor metastatic suppressor gene. However, it’s molecular mechanism of suppressing metastasis remains unexplored. There is a closely relationship between the abnormality of stucturs and functions of nm23-H1 gene, and cancer invasion and metastasis. We have constructed the vector with nm23-H1-shRNA and the vector with nm23-H1cDNA resistant to the specific shRNA. So, we plan to construct shRNA-resistant eukaryotic expression vector of nm23-H1 gene by site-directed mutagenesis, rescue experiment was performed to verify the nm23-H1 gene expression, and to provide basement for studying the biochemical mechanisms of nm23-H1 gene.
  
  
  METHODS
  Site-directed mutagenesis of nm23-H1 gene was performed by overlap extension PCR method. Pure plasmid containing gene of nm23-H1 (shRNA-resistant) was prepared. The desired five mutations were constructed and cloned into the eukaryotic vector pcDNA3.1Hygro(+). The human lung adenocarcinoma cell A549/nm23-H1-shRNA (stable nm23-H1 gene silencing) was transfected with the five mutants, and the expression of the mutant proteins was determined by Western blot.
  
  
  RESULTS
  Five eukaryotic expression vectors (shRNA-resistant) of nm23-H1, nm23-H1S44A, nm23-H1(P96S), nm23-H1(H118F), nm23-H1(S120G), nm23-H1(P96S-S120G), were successfully constructed. The results of DNA sequencing confirmed that the base sequences of the genes were completely concordant with experiment design. The expression of nm23-H1 mutant proteins was verified by Western blot.
  
  
  CONCLUSIONS
  Five eukaryotic expression vectors (shRNA-resistant) of nm23-H1 gene were successfully constructed, and the mutant proteins were verified. The site-directed mutagenesis technical of overlap extension PCR is a efficient, simple and economical method.
authors.affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China.
authors.forename: Zhansheng
authors.initials: Z
authors.lastname: Lu
authors.affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Guo
authors.affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Li
authors.affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China.
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Wu
authors.affiliation: Tianjin Key Laboratory of Lung Cancer Metastasis and Tumor Microenvironment, Tianjin Lung Cancer Institute, Tianjin Medical University General Hospital, Tianjin 300052, China.
authors.forename: Qinghua
authors.initials: Q
authors.lastname: Zhou
mesh_terms: D045744:Cell Line, Tumor; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D054778:NM23 Nucleoside Diphosphate Kinases / Q000235:genetics* / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism
chemical_list: D054778:NM23 Nucleoside Diphosphate Kinases; D034741:RNA, Small Interfering