新生大鼠脊髓损伤后大脑皮层cDNA文库中一条新基因的克隆及其相关蛋白功能的初步探讨

Identification of a novel gene Hs from the differential expression cDNA library and exploration of its protein function

目的从已构建的新生大鼠脊髓损伤后大脑皮层再生相关基因文库中克隆并鉴定一条再生相关基因H3（CA854305），并对其相关蛋白功能进行初步探讨。方法用Northern印迹技术观察新基因在体内存在及其表达变化。并用电子克隆（in silico cloning）技术对该基因进行全长克隆，用DNAstar对所获序列进行生物信息学分析，用RT—PCR技术对最长的开放阅读框（ORF）进行初步验证。结果Northern印迹显示新生大鼠脊髓损伤后5d，胚胎脊髓移植组对侧大脑皮层中的H3表达与损伤组和对照组比较，差异有统计学意义，移植组强于损伤组，损伤组强于对照组，提示H3可能与再生密切相关。电子克隆获得序列的最大长度为1635bp，最大ORF位于49—591bp，结构分析符合Cozak规则。RT—PCR实验证实最大ORF序列。结论新生大鼠皮层神经元轴突中断后的再生过程中有H3基因表达增强，可能与神经元再生有关。

Objective To clone and identify one novel regeneration related gene H3 （CA854305） from the differential expression genes library we had set up before and discuss its protein function. Methods Northern blotting was used to detect the different expressions of the novel gene under different situations. At the same time, the techniques of in silico cloning was employed to scan the span of H3 , the DNASTARTM software and OMIGATM software Ver4. 0. 5 to analyze their sequences, and reverse transcription PCR to validate the largest open reading frame （ORF）. Results Northern blotting results of H3 （CA854305） showed that transplantion group had efficiently more extensive expression than control and injured groups five days after spinal cord injury, with no statistical difference. While the expression of H3 in the transplantation group was more extensive than that in injured group, which was more extensive than control group. The results indicted that H3 might have some relationship with the regeneration after spinal cord injury. In silico cloning results got a longest contig of 1635 bp and an largest ORF of 542 bp from 49 -591 bp, which accorded with the Cozak rules. Reverse transcription PCR validated the largest ORF sequence primarily. Conclusions There exist increased expression of H3 gene during regeneration after cortical axonotmesis of neonate rats, which may correlate with nerve regeneration.