Cloning And Expression Of Bone Morphological Protein-7 (BMP-7) In Rabbit

The present thesis focuses on structural analysis of genes of the rabbit bone morphological protein-7(BMP-7),which may be offer information for researching embryo growth and roles in generation.We have obteained as follows results:1) We successfully isolated the most part of cDNA sequence of BMP-7 gene from rabbit kidney by 3'rapid amplification of cDNA ends and RT-PCR.The cDNA was 1654 bp (GenBank:EU004072;EF583950;EU599645) in length which contained the near full coding cDNA for the propetide,full coding cDNA for the mature protein,and 446 bp in length,the full length of 3'UTR of rabbit BMP-7.417 bp encoding the mature protein protein of 139 amino acids with a molecular mass of 15.6 kDa and an isoeletric point(pI) of 8.04.2) Sequence comparisons revealed that the full length of coding cDNA(1205 bp)of rabbit BMP-7 was 91.89%or 89.32%identical to that of human or mouse,while the deduced amino acids was 96.51%or 96.01%identical,respectively.The 3'UTR of rabbit BMP-7 was 446 bp in length,50.30%,41.45%,47.84%or 57.91%identical to that of human, mouse,cow or pig,respectively.In addition,there were two closely arranged AATAAA sites in the 3'UTR of rabbit BMP-7 gene,as well as in human BMP-7.The deduced mature protein of rabbit BMP-7 had the seven conservative cysteines and a TGF-βfamily signature,which were the characters of all BMPs.Our results suggested that the main part of rabbit BMP-7 has been successively cloned.The alternation of polyadenization sites in the 3'UTR of rabbit BMP-7 maybe related to the posttranscriptional regulation of the gene.3) The rabbit BMP-7 mRNA were expressed in many tissues and organs including liver, kidney,brain,spinal cord,lung,small intestine,spleen and heart muscle.But the mRNA expressiones of BMP-7 are different in different tissues.The expression in liver,kidney, brain are more than in other five tissues;the expression in spleen and heart muscle are the less.4) The successive construction of pET32a-BMP-7/BL21(DE3) and pET32a-BMP-7/BL21(DE3)PlysS supply good materials for the successive studies.The fused protein is 33.2 kDa which contained 15.6 kDa the mature protein of the rabbit BMP-7 and 17.6 kDa the protein of pET32a.The results show that under the conditions of 20℃～25℃,pET32a-BMP-7/BL21(DE3) induced by 0.2mM IPTG for 8h,or at 25℃～30℃,induced by 0.1mM IPTG for 5h may be procure more BMP-7.