In this study an exons connecting method was used to amplify the complete sequence encoding the mature peptide of endo-1,4-glucanase II of Trichoderma reesei QM9414. The gene was inserted into the Pichia pastoris vector pPIC9K, downstream of α-factor signal peptide sequence.
采用外显子拼接的方法,以里氏木霉Trichoderma reesei基因组 DNA 为模板,克隆出内切-1,4-β-D-葡聚糖酶II基因egl2的全编码序列,将其插入巴斯德毕赤酵母Pichia pastoris表达载体pPIC9K中,并位于α-因子信号肽序列的下游,获得重组质粒pQY2025。
Methods: The mature peptide of Apolipoprotein CⅡ is encoded by only two exons ,exon 3 and exon 4. The two exons were amplified respectively ,then they were linked together with the exons-connecting technique to obtain the DNA sequence encoding the mature peptide of ApoCⅡ,which is a 246bp DNA fragment.
METHODS: The two exons of SV40T were amplified respectively by high fidelity polymerase chain reaction (PCR) by using the plasmid pUC19-SV40T as the template. Then SV40T gene was spliced by overlapping extension (SOE), and cloned into the EcoR Ⅰ and BamH Ⅰ sites of the retroviral vector pLXSN.
In the previous work, we cloned SH2A Gene in this region by exon trapping and exon linking, which is a novel member of SH2 family. SH2 domain is considered as " Protein Recognized Code".
But now,with the deep research of functions of intron and genetic sequence,we find that the base alteration taken place in intron may cause the splice site change and produce dysfunction codogenic protein.
Objective Cloning and characterization of a novel gene by exon trapping and exon linking at chromosome 8p22. Methods A novel gene was cloned using exon trapping and exon linking, and its expression was determined by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot.