中国人肠道产甲酸草酸杆菌oxc基因的克隆及其在真核细胞中的表达

The cloning and identification of oxc gene from Oxalobacter formigenes in the intestines of Chinese people

目的观察中国人肠道产甲酸草酸杆菌（Ox．F）草酰辅酶A脱羧酶基因（oxc）的分离、克隆及其在293细胞中的表达。方法提取中国人肠道Ox．F的基因组DNA，PCR扩增oxc基因片段并克隆人真核表达载体pEGFP—C1，通过限制性内切酶酶切电泳和测序鉴定基因片段。将重组质粒脂质体转染至293细胞，利用RT-PCR和Western blot分别从mRNA和蛋白水平检测oxc基因在真核细胞中的表达。结果中国人肠道产甲酸草酸杆菌oxc基因全长1707bp，与Gene Bank中的序列比较，碱基序列的同源性为93．61％，氨基酸残基序列的同源性为97．18％。重组质粒转染293细胞后24～48h，可观察到明亮的绿色荧光，从mRNA和蛋白水平上可以检测到oxc基因在真核细胞中的表达。结论中国人肠道产甲酸草酸杆菌中可以分离出oxc基因；中国人肠道产甲酸草酸杆菌oxc基因存在一定的变异；oxc基因可在真核细胞293细胞中表达。

Objective To study the cloning and identification of oxc gene from Oxalobacter formigenes in the intestines of Chinese people. Methods The genomic DNA of Oxalobacter formigenes was extracted from the intestines of Chinese people. The fragment of oxc gene was amplified by polymerase chain reaction and linked with eukaryotic expression vector pEGFP-C1. The fragment of oxc gene was identified by restriction-enzyme cutting and sequencing. Human embryo kidney 293 cells were transfected with the recombinated plasmid by LipofectamineTM 2000. RT-PCR was performed to detect the mRNA expression of oxc. Western blot was used to detect the fusion protein expression of OCoAD-EGFP. Results The total length of oxc gene from Oxalobacter formigenes in the intestines of Chinese people was 1707 bp,and there were mutation of 109 nucleotides and 16 amino-acid residues. The homology of nucleotides and amino-acid residues with the sequence in GenBank was 93.61% and 97.18% respectively. The 293 cells transfected with the recombinated plasmid expressed relucent green fluorescence. The mRNA of oxc gene was detected by RT-PCR with a 347 bp amplified product. The fusion protein expression of OCoAD-EGFP was detected by Western blot with a 90kD protein. Conclusion The oxc gene can be cloned from the Oxalobacter formigenes in the intestines of Chinese people and there are some mutations with the oxc gene. The mutation oxc gene can be expressed in eucaryotic cells.