Cloning Of Human Noggin CDNA And Construction Of Mammalian Expression Vector PCS2+[Tα1]-Noggin

Objective Noggin was originally isolated from Xenopus embryos by Harland R.M. and Smith W.C. in 1992 . It was named as noggin because when they injected it into ventralized embryos at high doses , it caused excessive head development .Subsequently , people found that noggin plays an important role in normal development, especially of inducing dorsal development. Noggin is involved in numerous development processes , such as neural tube fusion and joint formation . The amino acid sequence of human noggin is highly homologous to that of Xenopus , rat and mouse . As a endogenous neural inducing signal ,noggin not only can be found in early vertebrate embryogenesis but also in the central neural system (CNS) and peripheral neural system (PNS) of adult mammalian . Noggin has been identified to be the neural inducer in vivo and in vitro , which effect neural induction through antagonizing bone morphogenetic protein-4(BMP4) . Up to now , there are many reports about neural differentiation in vitro both domestic and abroad , no one has constructed a recombinant vector with human noggin and induced neural cells with noggin.The sequence of the T alpha 1 gene contains the sequence elements responsible for specifying gene express to embryonic neurons and for subsequently regulating gene expression in both developing and mature neurons as a function of morphological growth. The present study was designed to constructed a vector of pCS2+[Tα1]-Noggin , thus providing an important and convenient tool to study the activity of noggin in vivo , such as gene expression ,protein localization , signal transformation , metabolic activity . The vector will be the foundation of the next research on the transfect of cells . The result will promote the farther research in the molecular biology of noggin gene , and the pCS2+[Tα1]-Noggin can be potentially used in cerebral disease gene therapy .Methods From the brain tissue of a normal human fetal , noggin cDNA fragment was amplified by reverse transcription-polymerase chain reaction (RT-PCR) , and cloned into pMD18-T plasmid . After it was confirmed by Hindlll and Xbal digest, we sent it to the corporation for sequencing . Then the product of PCR was digested with restrictive endonucleases Hind III and Xba I respectively , followed by ligation with pCS2+[Tal]-GFP vector which had been digested with Hindlll and Xbal . The E.coli strain DH5a was transformed with the mixture of ligation . Then transformation colonies were picked at random, and the recombinant plasmid were extracted . Having been verified via restrictive endonucleases digest, PCR and DNA sequencing . Then pCS2+[Tal]vector of noggin was constructed .Result Analysis of the sequence results showed that the full length of the fragment cloned from brain tissue of human fetal was correct amplified .This sequence is coincident with the human noggin cDNA sequence in the GenBank(accession number: NM₀05450) as expected. Later, the recombinant plasmid has been successfully constructed .Conclusion In this study we cloned noggin gene from brain of human fetal and constructed recombinant mammalian expression vector pCS2+[Tal]-Noggin.