Clone Of Heat-shock Protein Of Crabs And The Expression Research Of Different Tissue

Heat shock protein 70kDa (HSP70) is one of the most important proteins among members of HSPs, and mainly functions as molecular chaperone. Moreover, HSP70s play fundamental roles in cytoprotection, anti-apoptotic effects, and immune therapy of tumor, etc. Therefore, researches on HSP70s have become one of popular and prospective focuses in fields of life science.Firstly, by the study of the HSP70 gene of Callinectes sapidus and Litopenaeus vannamei, according to their sequences, I designed the proper primers and cloned a HSP70 gene from the ovum tissue after 38℃heat-stressed Eriochier sinensis by using RT-PCR and RACE. The cDNA sequence was 2429 bp in length and encoded a 649 (Mr 70.9kDa) amino-acid polypeptide with isoelectric point (pI) 5.3. Based on the phylogenic tree of HSP70s, it indicated that it belonged to the member of constitutive HSC70 family with high identities (66-91%) of amino-acid sequence with other 29 known HSP70s.In the same way, a HSP70 gene was cloned from ovum tissue of Scylla serrata. This HSP70 was 2482bp in length and encoded a 649 (Mr 71.06kDa) amino-acid polypeptide with pI 5.24. It includes a 1950bps open reading frame. There was a polyadenylation signal of AATAAA found in 3' end of the sequence. Based on the phylogenic tree of HSP70s, this HSP70 also belonged to the constitutive HSC70 family.Semi-quantitative RT-PCR was developed to study the HSC70 transcription pattern in E. sinensis and S. serrata. The level of HSC70 mRNA is constitutively express in ovum, heart, muscle, stomach, intestines of non-stressed E. sinensis. After heat shock in 38℃water, the levels of HSC70 mRNA have up-regulated. The highest expression of HSC70 of E. sinensis was observed in ovum and heart organ. The result confirmed that this gene belonged to the constitutive HSC70 family.The RT-PCR assay demonstrated that HSC70 of S. serrata constitutively expressed in ovum, heart, muscle, stomach and intestines. After heat shock in 38℃water, the levels of HSC70 mRNA have up-regulated. The highest expression of HSC70 of S. serrata was observed in ovum. The result also suggested that this gene belonged to the constitutive HSC70 family.