Polypeptide-human X linked gene espression protein 14 and polynucleotide for encoding such polypeptide
A technology for polynucleotide and gene expression, applied in the field of preparation of polynucleotides and polypeptides
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Embodiment 1
[0121] Total RNA was extracted from human fetal brain by one-step method of guanidine isothiocyanate / phenol / chloroform. Poly(A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (product of Qiegene). 2ug poly(A) mRNA was reverse transcribed to form cDNA. Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragment into the multiple cloning site of the pBSK(+) vector (product of Clontech Company), transform DH5α, and the bacteria formed a cDNA library. The sequences of the 5' and 3' ends of all clones were determined using Dyeterminate cycle reaction sequencing kit (product of Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer). Comparing the determined cDNA sequence with the existing public DNA sequence database (Genebank), it was found that the cDNA sequence of one of the clones, 1073A09, was a new DNA. The insert cDNA fragment contained in this clone was determined bidirectionally by synthesizing a series of primers. The resu...
Embodiment 2
[0122] Example 2: Homology retrieval of cDNA clones
[0123] The sequence of the human X-linked gene expression protein 14 of the present invention and the protein sequence encoded by it, use the Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10] , perform homology search in Genbank, Swissport and other databases. The gene with the highest homology to the human X-linked gene expression protein 14 of the present invention is a protein expressed by a known X-linked gene Bex2, and the accession number of the encoded protein in Genbank is AF097439. The results of protein homology are shown in Figure 1. The two are highly homologous, with an identity of 71% and a similarity of 85%. Embodiment 3: Cloning the gene encoding human X-linked gene expression protein 14 by RT-PCR method
Embodiment 3
[0123] The sequence of the human X-linked gene expression protein 14 of the present invention and the protein sequence encoded by it, use the Blast program (Basiclocal Alignment search tool) [Altschul, SF et al.J.Mol.Biol.1990; 215:403-10] , perform homology search in Genbank, Swissport and other databases. The gene with the highest homology to the human X-linked gene expression protein 14 of the present invention is a protein expressed by a known X-linked gene Bex2, and the accession number of the encoded protein in Genbank is AF097439. The results of protein homology are shown in Figure 1. The two are highly homologous, with an identity of 71% and a similarity of 85%. Embodiment 3: Cloning the gene encoding human X-linked gene expression protein 14 by RT-PCR method
[0124] The total RNA of fetal brain cells was used as a template, and oligo-dT was used as a primer to carry out reverse transcription reaction to synthesize cDNA. After purification with a Qiagene kit, PCR amp...
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