【作者】 孙勇；

【导师】 谢数涛；

【作者基本信息】 暨南大学 ， 水生生物学， 2006， 硕士

【摘要】 热休克蛋白70(heat shock protein 70，HSP70)是热休克蛋白家族中最重要的一类家族成员，它具有“分子伴侣”、细胞保护、抗凋亡以及肿瘤免疫治疗等独特复杂的生物学功能，因此，它的研究已成为生命科学领域研究的热点之一。 本研究首先电子克隆了蓝蟹(Callinectes sapidus)HSC70基因cDNA的全长序列以及日本对虾(Marsupenaeus japonicus)HSP70部分序列，设计引物，采用RT-PCR和RACE(rapid amplification of cDNA ends)相结合的方法，从35℃热休克的克氏原螯虾(Procambarus Clarkii)的心脏组织克隆得到长2271bp的HSP70基因(命名为Pro-HSP70基因)，编码635个氨基酸的多肽分子，推测的分子量为70kDa，理论等电点为5.34。经基因组DNA扩增，发现该基因不含内含子。与已知的29个其它生物的HSP70或HSC70蛋白序列的同源性为68.4～86.3％，通过构建系统发生树，发现Pro-HSP70可能是一种诱导型HSP70基因。 从未经热休克的刀额新对虾(Metapenaeus ensis)的心脏组织中克隆得到长2242bp的HSC70(heat shock cognate 70)基因(命名为Met-HSC70基因)，包括长1947bp的完整编码区，在poly(A)尾上游发现一加尾信号(AATAAA)。编码648个氨基酸的多肽分子，分子量为71kDa，理论等电点为5.06。经基因组DNA扩增，发现该基因含有内含子。与已知的29个其它生物的HSP70或HSC70蛋白序列构建系统发生树，发现Met-HSC70可能是一种组成型HSP70基因。 从35℃热休克的日本沼虾(Macrobrachium nipponense)的心脏组织克隆得到长2553bp的HSP70基因(命名为Mac-HSC70基因)，编码649个氨基酸的多肽分子，推测的分子量为71kDa，理论等电点为5.04。通过序列分析构建系统发生树，发现Mac-HSC70可能是一种组成型HSP70基因。 采用半定量RT-PCR方法，对两种虾的HSP70组织表达进行研究。克氏原螯虾的HSP70 mRNA正常情况下在心脏、肌肉、血液、消化腺、触角腺、精巢和肠组织中表达量相当低下，但是经过35℃、2h热休克以后，表达量显著升高，进一步说明Pro-HSP70是一种热诱导型的热休克蛋白，其中心脏的表达量为最高。在1～8h的热应激实验中，心脏和触角腺Pro-HSP70 mRNA的变化呈上升趋势。非应激状态下，刀额新对虾在心脏、肝胰腺、鳃、脑、胃、精巢和肠中均大量表达HSC70 mRNA，表明克隆得到的Met-HSC70为组成型HSC70基因。更多还原

【Abstract】 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更多还原