【作者】 王红艳；

【导师】 肖蓉；

【作者基本信息】 辽宁师范大学 ， 海洋生物学， 2015， 硕士

【摘要】 组织蛋白酶D(cathepsin D,CTSD)是存在于溶酶体内的一种天冬氨酸类蛋白质水解酶,主要参与蛋白质的水解等过程。七鳃鳗是低等无颌类动物,通常以鱼类的血肉为生。前期结果表明七鳃鳗的口腔腺中含有组织蛋白酶D基因(Lamprey-cathepsin D,L-CTSD),但是截止到目前为止还未有任何研究报道L-CTSD在七鳃鳗中所发挥的作用。为了深入研究L-CTSD的生物学功能,本研究构建了L-CTSD的原核表达载体pColdⅠ-L-CTSD,并将其转化至大肠杆菌Rosetta中。电泳结果显示重组的七鳃鳗CTSD(recombinant lamprey CTSD,rL-CTSD)以包涵体形式表达。采用变性及复性的方法得到了分子量约为43 kDa电泳纯度的可溶性rL-CTSD。研究还构建了L-CTSD的真核表达载体pcDNA3.1-L-CTSD,并经脂质体将其转染至293T细胞中。采用亲和层析纯化后得到了可溶性的rL-CTSD。重组蛋白质经Western Blot及质谱鉴定后确定为rL-CTSD。功能试验结果显示无论是通过原核还是真核表达的rL-CTSD均能够在酸性条件下降解血液中的主要成分,如血红蛋白、纤维蛋白原和血清白蛋白。这表明口腔腺中的L-CTSD参与了七鳃鳗的进食消化过程。此外,这两种CTSD还能抑制大肠杆菌的生长,表明其还能够参与调节七鳃鳗的免疫防御过程。因此,对L-CTSD的重组表达及功能研究可为后续深入阐明七鳃鳗的进食机制奠定基础,为今后保护七鳃鳗资源提供数据支撑。此外,七鳃鳗口腔腺中还含有丰度较高的富含半胱氨酸分泌蛋白家族成员,即富含半胱氨酸口腔腺分泌蛋白(Cysteine-rich buccal gland protein,CRBGP)。该蛋白质由PR-1(pathogenesis-related group 1 domain)和CRD(cysteine-rich domain)结构域组成。前期结果表明CRBGP具有多样的生物学功能,在离子通道调控和抗血管新生等方面发挥重要作用。为了深入阐明CRBGP蛋白质中PR-1结构域和CRD结构域与其功能的关系,本研究还首次构建了pET42a-L-PR-1和pET42a-L-CRD的原核表达载体,并成功表达出分子量分别为52 kDa和39 kDa的可溶性重组蛋白:PR-1结构域(recombinant L-PR-1,rL-PR-1)和CRD结构域(recombinant L-CRD,rL-CRD)。MTT实验结果表明:rL-PR-1和rL-CRD均能抑制人脐静脉血管内皮细胞(Human Umbilical Vein Endothelial Cells,HUVEC)的增殖,不过rL-CRD对HUVEC细胞增殖的抑制作用更为明显,提示CRD结构域抑制在HUVEC的增殖过程中起主要作用。rL-PR-1和rL-CRD的重组表达可为后续深入研究其功能奠定基础。更多还原

【Abstract】 Cathepsin D(CTSD)is an aspartic proteinase which is present in the lysosome and plays important roles in the process of protein degradation.The primitive jawless lampreys usually live on the blood and flesh of host fish.The previous studies have shown that cathepsin D(Lamprey-cathepsin D,L-CTSD)is expressed in the buccal gland of lampreys.However,the functions of L-CTSD have not been reported yet.In order to study the functions of L-CTSD,pColdⅠ-L-CTSD was constructed and successfully expressed in Escherichia coli Rosetta.SDS-PAGE showed that recombinant L-CTSD(rL-CTSD)was expressed as the inclusion bodies.The soluble protein with the molecular weight of 43 kDa was obtained and migrated as a single band after denaturation and renaturation.In addition,pcDNA3.1-L-CTSD was also successfully constructed and transfected in 293 T cells by liposome.The soluble rL-CTSD was got through affinity chromatography.The recombinant protein was identified as rL-CTSD by using Western Blot and mass spectrometry.Both expressed rL-CTSD could degrade hemoglobin,fibrinogen and serum alumin in acidic conditions,which are the main protein component of blood.This suggests that L-CTSD plays important roles in the feeding and digestion processes of lampreys.In addition,the proliferation of Escherichia coli could be inhibited by both rL-CTSD,which indicates L-CTSD participates in the immune response of lampreys.Thus,the expression and functions studies of L-CTSD will not only help to illuminate the feeding mechanism of lampreys,but also protect them from death.Furthermore,the lamprey buccal glands also contain cysteine-rich secretory protein family member: cysteine-rich buccal gland protein(CRBGP),which is composed of a pathogenesis-related group 1 domain(PR-1)and a cysteine-rich domain(CRD).Previous studies have shown that CRBGP plays important roles in the regulation ion channels and has the anti-angiogenic activity.In order to illuminate the relationship between functions of CRBGP and its PR-1 and CRD domains,pET42a-L-PR-1 and pET42a-L-CRD were successfully constructed.The recombinant L-PR-1(rL-PR-1)and L-CRD(rL-CRD)were expressed as soluble proteins with the molecular weight of 52 kDa and 39 kDa,respectively.According to the assay of MTT,both rL-PR-1 and rL-CRD could inhibit the proliferation of human umbilical vein endothelial cells.In addition,the inhibition effect induced by rL-CRD was more strong than that of rL-PR-1,suggested that CRD domain plays important roles in L-CTSD was up-regulated in the above tissues,which suggested that L-CTSD may play the inhibition of HUVEC proliferation,Thus,the expression of PR-1 and CRD domain provide the basis for further studies of the functions of CRBGP.更多还原