Cloning And Expression Of HSP70 And HSC70 Genes From Three Shrimps: Procambarus Clarkii, Metapenaeus Ensis And Macrobrachium Nipponense

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.In our present study, we selected Procambarus Clarkii, which is the commercially important organism, as the experimental material. Firstly, The whole Callinectes sapidus HSP70 sequence and partial Marsupenaeus japonicus HSP70 sequence were silico cloned on the basis of EST database. According to them, we designed specific primers and cloned a HSP70 gene from heart organ of 35℃ heat-stressed P. Clarkii using RT-PCR and RACE method. The cDNA sequence, which named Pro-HSP70, was 2271 bp in length and encoded a 635 (Mr 70kDa) amino-acid polypeptide with isoelectric point (pI) 5.34. Genomic DNA sequence amplification revealed that Pro-HSP70 has no introns. Based on the phylogenic tree of HSP70s, it indicated that it belonged to the member of inducible HSP70 family with high identities (68.4-86.3%) of amino-acid sequence with other 29 known HSP70s.With the same methods, a HSC70 gene was cloned from heart organ of non heat-stressed Metapenaeus ensis and was named Met-HSC70. Met-HSC70 was 2242bp in length and encoded a 648 (Mr 71kDa) amino-acid polypeptide with pI 5.06. Genomic DNA sequence amplification revealed that Met-HSC70 has introns.There was a polyadenylation signal found in 3' end of the sequence. Based on the phylogenic tree of HSP70s, Met-HSC70 belonged to the constitutive HSC70 family.With the same methods, we cloned a HSC70 gene from heart organ of 35℃ heat-stressed Macrobrachium nipponense. The cDNA sequence, which named Mac-HSP70, was 2553 bp in length and encoded a 649 (Mr 71kDa) amino-acid polypeptide with isoelectric point (pI) 5.04. Based on the phylogenic tree of HSP70s with other 29 known HSP70s' amino-acid sequences, it indicated that Mac-HSP70belonged to the member of the constitutive HSC70 family.Semi-quantitative RT-PCR was developed to study the HSP70 or HSC70...