Polypeptide-human transcription regulate and control protein 44 and polynucleotide for coding said polypeptide
A polynucleotide and transcriptional regulation technology, applied in the field of polynucleotide and polypeptide preparation, can solve problems such as weak activity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0080] Example 1: Cloning of human transcriptional regulatory protein 44
[0081] The total RNA of human fetal brain was extracted with guanidine isothiocyanate / phenol / chloroform one-step method. Quik mRNA Isolation Kit (product of Qiegene) was used to isolate poly(A)mRNA from total RNA. 2ug poly(A)mRNA is reverse transcribed to form cDNA. Use Smart cDNA cloning kit (purchased from Clontech) to insert cDNA fragments into the multiple cloning site of pBSK(+) vector (product of Clontech), transform DH5α, and form cDNA library by bacteria. Dye terminate cycle reaction sequencing kit (product of Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) were used to determine the sequence of the 5'and 3'ends of all clones. The determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0426G01 was new DNA. By synthesizing a series of primers, the inserted cDNA fragments contained in t...
Embodiment 2
[0082] Example 2: Homologous search of cDNA clones
[0083] Use the Blast program (Basiclocal Alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10] to use the Blast program (Basiclocal Alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10]. Genbank, Swissport and other databases perform homologous search. The gene with the highest homology to the human transcriptional regulatory protein 44 of the present invention is a known human transcriptional regulatory protein 136, and the encoded protein has an access number of U09367 in Genbank. The results of protein homology are shown in Figure 1. The two are highly homologous, with an identity of 53% and a similarity of 67%.
Embodiment 3
[0084] Example 3: Using RT-PCR method to clone the gene encoding human transcriptional regulatory protein 44
[0085] Using total RNA from fetal brain cells as a template and oligo-dT as primers for reverse transcription reaction to synthesize cDNA, after purification with Qiagene kit, PCR amplification was performed with the following primers:
[0086] Primer1: 5’-ACGGCTGCGAGAAGACGAAGCTTAG-3’(10)
[0087] Primer2: 5’-AATTAATAACAATTCTTTATTAGC-3’(11)
[0088] Primer1 is the forward sequence starting from the 1st bp at the 5'end of 1;
[0089] Primer2 is the 3'reverse sequence in 1.
[0090] Amplification reaction conditions: 50mmol / L KCl, 10mmol / LTris-Cl, (pH8.5), 1.5mmol / L MgCl in a reaction volume of 50μl 2 , 200μmol / L dNTP, 10pmol primers, 1U Taq DNA polymerase (product of Clontech). The reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94°C 30sec; 55°C 30sec; 72°C 2min. In RT-PCR, β-actin was set as the positive c...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More