| Objective Cloning of human neurotrophin-4(hNT-4) gene and the strategy for constructing the recombinant pcDNA3.1(+)/hNT-4.
Methods With the chromosomal DNA of human blood lymphocytes as template, hNT-4 coding genes were amplified by polymerase chain reaction(PCR) and recombinated into phage vector pGEM-T Easy,which were sequenced by using Sanger’s single stranded DNA terminal termination method.Through genetic recombination technique, human neurotrophin-4 gene (hNT-4) was inserted into polylinker site of expression vector pBV220, to generate a recombination plasmid pBV220/ hNT-4 as expression vector. (hNT-4) was inserted into polylinker site of expression vector pcDNA3.1(+), to generate a recombination plasmid pcDNA3.1(+)/hNT-4 as expression vector.
Results The sequence of the cloned gene is completely the same as that reported in the literature (GenBank data base, M86528). The results showed that hNT-4 was cloned correctly into expression vector pcDNA3.1(+), recombinant expression vector pcDNA3.1(+)/hNT-4 was constructed. Open reading frame is not exchange.
Conclusion This study successfully cloning the gene of mhNT-4 and the strategy for constructing the recombinant pcDNA3.1(+)/hNT-4,and it would be convenient for us to study the expression of mhNT-4 in eukaryote, and to continue the research on the gene therapy of Alzheimer’s disease intensively. This study indicate that the hNT-4 is conservative in different races and individuals.
Key words human neurotrophin-4(hNT-4), neurotrophin-4（NT-4）,gene cloning, DNA sequencing, polymerase chain reaction(PCR)，pcDNA3.1(+) vector, pcDNA3.1(+)/hNT-4
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