Carboxin抗性基因(Cbx~R)原核表达及多克隆抗体的制备

[目的]克隆CbxR基因,进行原核表达,纯化CbxR基因蛋白并制备其多克隆抗体。[方法]CbxR基因克隆至原核表达载体p ET-28a(+),转化大肠杆菌BL21(DE3)诱导表达并纯化,Western blot鉴定分析。制备CbxR蛋白的兔源多克隆抗体,运用间接ELISA方法检测多抗效价,利用Western blot印记检测抗体特异性。[结果]成功获得CbxR基因,在大肠杆菌BL21(DE3)中诱导可大量表达,成功制备CbxR蛋白兔源多克隆抗体,效价为1∶64 000,经Western blot检测表明多克隆抗体特异性良好。[结论]多克隆抗体为CbxR检测及进一步研究CbxR基因功能奠定了基础。     

粗糙脉孢菌ERG-11蛋白抗原的原核表达、纯化及其多克隆抗体制备

构建p ET-28a(+)-ERG-11重组质粒,表达6×His-ERG-11融合蛋白,制备ERG-11多克隆抗体。采用PCR技术扩增目的片段,插入p ET-28a(+)原核表达载体,并转入E.coli BL21(DE3)感受态表达融合蛋白,融合蛋白经亲和纯化及分子筛纯化后免疫新西兰大白兔制备多克隆抗体,取血清后,采用间接ELISA法和Western blot法检测多克隆抗体的效价及特异性。成功构建了p ET-28a(+)-ERG-11表达载体,SDS-PAGE电泳显示成功诱导出以包涵体形式存在的6×His-ERG-11融合蛋白,两步纯化后得到纯度较高的抗原,间接ELISA法显示制备的多克隆抗体效价达到1∶512 000,Western blot显示具有较高特异性。成功实现了粗超脉孢菌ERG-11蛋白的原核表达,制备出一支兔抗粗超脉孢菌ERG-11的多克隆抗体。     

人WWP2蛋白的重组表达和多克隆抗体的制备

[目的]克隆、表达并纯化人E3泛素连接酶蛋白WWP2的部分肽段(aa 324-517),制备兔源抗WWP2多克隆抗体并初步鉴定。[方法]PCR法从人胚肾细胞HEK-293T中扩增WWP2蛋白部分肽段的编码序列并构建原核表达质粒,大肠杆菌中诱导表达;GST亲和层析法纯化后进行TEV酶切,免疫新西兰兔制备多克隆抗体;间接ELISA、Western Blot、免疫荧光等方法检测抗体的灵敏度和特异性。[结果]构建了原核表达质粒pGEX-GST-WWP2(aa 324-517),原核表达并纯化了该重组蛋白。间接ELISA测定抗体效价可达1∶100 000以上,Western Blot检测该抗体可特异性识别体外纯化的WWP2蛋白和细胞内源性WWP2蛋白,细胞免疫荧光可检测到内源性WWP2蛋白。[结论]成功克隆、表达与纯化WWP2蛋白的部分肽段,制备出抗WWP2蛋白的多克隆抗体,可用于WWP2的免疫印迹和细胞免疫荧光分析。     

拟南芥转录因子NF-YC的原核表达及多克隆抗体制备

采用PCR方法扩增NF-YC基因得到其全长cDNA序列,并将其克隆至原核表达载体pET-48b中,在大肠杆菌BL21中用IPTG诱导出分子量约为45 kD的融合蛋白,SDS-PAGE和Western blotting检测鉴定表达产物。利用亲和层析技术对融合蛋白进行纯化,纯化后的目的蛋白免疫新西兰兔制备多克隆抗体。间接ELISA检测抗体效价大于1 62 500,Western blotting结果显示,该抗体可特异性识别NF-YC蛋白。     

人接头蛋白NRBP的表达、纯化及多克隆抗体制备

目的:制备接头蛋白NRBP的兔多克隆抗体,并检测该抗体的效价及特异性。方法:PCR方法以重组质粒PEF-NRBP为模板,获得NRBP全长及NRBP(1-99Aa)cDNA,构建到原核表达质粒pET-21a及pGEX-6P-1中。分别转入大肠杆菌BL21菌株,IPTG诱导表达后,纯化并鉴定表达产物将其免疫家兔,间接ELISA法及免疫印迹等方法鉴定抗体。结果:成功获得人NRBP的cDNA,构建得到相关的原核表达质粒,在大肠杆菌中可诱导性高表达,纯化后蛋白免疫家兔制备得到的抗血清经ELISA检测为阳性结果,4只免疫家兔的抗体效价约为1:5 200～1:40 000。Western印迹结果显示,该抗体可特异性的识别真核细胞外源性及内源性约60kDa的NRBP蛋白,并且具有较强免疫沉淀能力。结论:NRBP多克隆抗体具有很好的特异性和敏感性,该抗体的成功制备为进一步研究NRBP的功能提供了重要工具。     

茶树咖啡碱合成酶基因原核表达、及其抗体制备与鉴定

克隆出茶树咖啡碱合成酶基因,对其进行原核表达,并制备TCS1抗体,旨在从蛋白水平研究茶树体内TCS1的表达情况。根据Gen Bank登陆的TCS1基因的全长c DNA序列,找出其完整的ORF(开放阅读框),从茶树叶片c DNA中克隆了TCS1基因的开放阅读框,连接到p GEX-4T-2表达载体,经IPTG诱导表达重组蛋白p GEX-4T-2-TCS1。进行体外酶活检测后,亲和层析纯化重组蛋白,作为抗原免疫家兔,制备TCS1多克隆抗体。用ELISA方法检测抗体效价,Western blot检测抗体的特异性。通过优化诱导条件,得出重组蛋白的最佳表达条件为:30℃、4 h。诱导后的总蛋白、可溶性蛋白与包涵体蛋白均出现一条明显的外源蛋白条带。抗体经ELISA检测,效价为1∶2 000,Western blot检测表明抗体具有相对较好的特异性。构建了TCS1原核表达质粒,同时成功制备了抗TCS1的多克隆抗体。     

重组丙型肝炎病毒核心蛋白的原核表达、纯化和抗体制备

本文旨在获得纯化丙型肝炎病毒(HCV)核心蛋白(HCV-C)及抗HCV-C多克隆抗体,为深入研究HCV-C与肝细胞相互作用的分子机制奠定基础。首先以HCV1b亚型HC-J4-91全基因组质粒为模板,聚合酶链反应(PCR)扩增HCV-C基因,构建重组质粒pQE31-HCV-C。融合蛋白经原核表达、纯化后,免疫BALB/c小鼠,制备抗HCV-C多克隆抗体。利用酶联免疫吸附试验(ELISA)检测抗体效价,蛋白免疫印迹(Westernblot)和间接免疫荧光染色鉴定抗体特异性。结果显示,表达HCV-C的原核表达质粒pQE31-HCV-C构建正确,获得相对分子质量约22000的纯化融合蛋白。ELISA检测重组蛋白免疫小鼠的抗血清效价达1:12800。结果显示,自制的抗HCV-C多克隆抗体能特异性识别HCV-C。本研究获得了纯度较好、原核表达的HCV-C,并成功制备了抗HCV-C多克隆抗体,为深入研究HCV-C的致病机制提供了有实用价值的研究工具。     

DNA损伤检查点蛋白调节子1片段的表达纯化及抗体制备

目的：原核表达、纯化DNA损伤检查点蛋白调节子1（MDC1）片段,并制备其多克隆抗体。方法：设计特异引物,通过RT-PCR扩增编码MDC1 N端194个氨基酸残基的基因片段,测序正确后插入含GST基因的原核表达载体pGEX-KG中,以IPTG诱导表达,并经谷胱甘肽琼脂糖珠纯化融合蛋白;用纯化的蛋白免疫小鼠制备多克隆抗体,用ELISA测定抗体的效价,Western印迹鉴定抗体的特异性。结果：原核表达并纯化了MDC1 N端片段,并获得了抗MDC1的多克隆抗体,抗体效价达到1∶12800,Western印迹显示该抗血清能特异识别原核及真核细胞表达的MDC1。结论：MDC1 N端片段能够诱导小鼠产生具有较高效价和特异性的多克隆抗体,为进一步研究MDC1在Fhit特异信号通路中的作用奠定了基础。     

美洲大蠊i型溶菌酶的原核表达及多克隆抗体制备

旨在获得美洲大蠊i型溶菌酶Pa I的原核表达蛋白,并制备鼠抗Pa I多克隆抗体。PCR扩增Pa I成熟肽编码序列,构建原核表达载体p ET28a-Pa I,诱导表达、纯化Pa I-His融合蛋白,免疫Balb/c小鼠,间接ELISA法检测抗血清效价,Western blotting检测多克隆抗体的特异性。结果显示,Pa I成熟肽部分的编码序列长414 bp,由137个氨基酸组成。构建的原核表达载体p ET28a-Pa I在大肠杆菌Rosetta(DE3)中成功表达,SDS-PAGE检测显示纯化的Pa I-His融合蛋白的大小约为18 k D,间接ELISA和Western blotting分析表明免疫小鼠产生的抗血清具有较高的效价和较强的特异性。美洲大蠊溶菌酶Pa I成功实现原核表达,并获得了高效价特异性多克隆抗体。     

钼还原菌中TrxR的原核表达及多克隆抗体制备

[目的]构建枯草芽孢杆菌硫氧还蛋白还原酶(thioredoxin reductase,TrxR)原核载体,表达、纯化TrxR重组蛋白,制备并鉴定多克隆抗体。[方法]通过分子克隆获得TrxR蛋白的表达菌株;利用镍离子亲和层析获得纯化的TrxR重组蛋白,免疫兔子制备TrxR蛋白多克隆抗体;采用ELISA法测定抗体效价;Western Blot检测抗血清的特异性。[结果]TrxR重组载体双酶切结果与DNA测序鉴定结果一致,蛋白表达纯化条带大小与预测一致。ELISA法测定抗血清效价为7×104,Western Blot证实抗血清有较高的特异性。[结论]成功克隆、表达与纯化TrxR重组蛋白,制备并鉴定兔子多克隆抗体,为TrxR的生物学功能研究奠定基础。     

Study of DFF45 in its role of chaperone and inhibitor: two independent inhibitory domains of DFF40 nuclease activity.

CAD/DFF40, the nuclease responsible for DNA fragmentation during apoptosis, exists as a heterodimeric complex with DFF45/ICAD. This study determines the molecular mechanisms of regulation of DFF40 via the chaperone and inhibition activities of DFF45. We analyze proteins corresponding to the fragments (D1, D2, and D3) of DFF45 generated by cleavage at the caspase consensus sites in DFF45. Either D1 or D2, as an isolated domain, is capable of inhibiting DFF40 nuclease activity while double domain fragments D1-2 and D2-3, as well as full-length DFF45, bind to DFF40 with high affinity and are much more effective inhibitors. The chaperone activity of DFF45 resides in part in its ability to maintain DFF40 as a soluble protein. In addition, D1 of DFF45 was found to be critical for the expression of active DFF40 in vivo, suggesting a role for DFF45 in binding nascent DFF40.     

A new method for protein coexpression in Escherichia coli using two incompatible plasmids

It is commonly believed that incompatible plasmids carrying the same replicon cannot coexist stably in one Escherichia coli cell. However, we found that two incompatible plasmids carrying different antibiotic resistance genes, if under the selection pressure of the two antibiotics, can coexist in E. coli for at least 14 h, which is adequate for routine culture and protein expression. Based on this discovery, we developed a new method to coexpress foreign proteins in E. coli using two incompatible plasmids. The coding regions of the two subunits (DFF45 and DFF40) of the human DNA fragmentation factor (DFF) were cloned into two incompatible bacterial expression vectors-pET-21a with ampicillin resistance and pET-28a with kanamycin resistance, respectively. The two resulting plasmids were used to cotransform E. coli BL21(DE3) cells. After selection by ampicillin and kanamycin simultaneously, cotransformants that contain both recombinant plasmids were obtained. Induced by isopropyl beta-d-thiogalactoside, DFF45, and DFF40 were coexpressed efficiently in the presence of the two antibiotics. The coexpression product contained adequate soluble portions for both DFF45 and DFF40, while all DFF40 was insoluble if expressed alone. The coexpression product also exhibited the same caspase-activated DNase activity as its natural counterparts, which cannot be obtained if its two subunits are expressed separately.     

Activation of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease). Oligomerization and direct interaction with histone H1.

DNA fragmentation factor (DFF) is a heterodimeric protein composed of 45-kDa (DFF45) and 40-kDa (DFF40) subunits, a protein that mediates regulated DNA fragmentation and chromatin condensation in response to apoptotic signals. DFF45 is a specific molecular chaperone and an inhibitor for the nuclease activity of DFF40. Previous studies have shown that upon cleavage of DFF45 by caspase-3, the nuclease activity of DFF40 is relieved of inhibition. Here we further investigate the mechanism of DFF40 activation. We demonstrate that DFF45 can also be cleaved and inactivated by caspase-7 but not by caspase-6 and caspase-8. The cleaved DFF45 fragments dissociate from DFF40, allowing DFF40 to oligomerize to form a large functional complex that cleaves DNA by introducing double strand breaks. Histone H1 directly interacts with DFF, confers DNA binding ability to DFF, and stimulates the nuclease activity of DFF40 by increasing its Kcat and decreasing its Km.     

The differences in RCAS1 and DFF45 endometrial expression between late proliferative, early secretory, and mid-secretory cycle phases

RCAS1 expression is related to the regulation of activated immune cells and to connective tissue remodeling within the endometrium. DFF45 seems to play an important role in the apoptotic process, most likely by acting through the regulation of DNA fragmentation. Its expression changes within the endometrium seem to be related to the resistance of endometrial cells to apoptosis. The aim of the present study was to evaluate RCAS1 and DFF45 endometrial expressions during ovulation and the implantation period. RCAS1 and DFF45 expression was assessed by the Western-blot method in endometrial tissue samples obtained from 20 patients. The tissue samples were classified according to the menstrual cycle phases in which they were collected, with a division into three phases: late proliferative, early secretory, and mid-secretory. The lowest level of RCAS1 and the highest level of DFF45 endometrial expression was found during the early secretory cycle phase. Statistically significantly higher RCAS1 and statistically significantly lower DFF45 endometrial expression was identified in the endometrium during the late proliferative as compared to the early secretory cycle phase. Moreover, statistically significantly higher RCAS1 and statistically significantly lower DFF45 expression was found in the endometrium during the mid-secretory as compared to the early secretory cycle phase. The preparation for implantation process in the endometrium is preceded by dynamic changes in endometrial ECM and results from the proper interaction between endometrial and immune cells. The course of this process is conditioned by the immunomodulating activity of endometrial cells and their resistance to immune-mediated apoptosis. These dynamic changes are closely related to RCAS1 and DFF45 expression alterations.     

Cell death-inducing DFF45-like effector, a lipid droplet-associated protein, might be involved in the differentiation of human adipocytes

Cell death-inducing DFF45-like effector (CIDE) family proteins, including cell death-inducing DFF45-like effector A (CIDEA), cell death-inducing DFF45-like effector B (CIDEB) and cell death-inducing DFF45-like effector C (CIDEC) [fat-specific protein of 27 kDa in rodent (FSP27) in rodents], were originally identified by their sequence homology to the N-terminal region of DNA fragmentation factor DFF40/45. Recent reports have revealed that CIDE family proteins play important roles in lipid metabolism. Several studies involving knockdown mice revealed that FSP27 is a lipid droplet-targeting protein that can promote the formation of lipid droplets. However, the detailed roles of human CIDEC in the differentiation of human adipocytes remain unknown. In the present study, we found that the expression of CIDEC increased during the differentiation of fetal adipose tissues, but decreased during the de-differentiation of adipocytic tumors, suggesting that the expression of CIDEC should be positively correlated with the differentiation of adipocytes. Furthermore, we verified that human CIDEC was localized on the surface of lipid droplets. Using human primary pre-adipocytes, we confirmed that the expression of CIDEC was elevated during the differentiation of pre-adipocytes, and knockdown of CIDEC in human primary pre-adipocytes resulted in differentiation defects. These data demonstrate that CIDEC is essential for the differentiation of adipose tissue. Together with regulating adipocyte lipid metabolism, CIDEC should be a potential target for regulating adipocyte differentiation and reducing fat cell mass.     

Generation of aberrant forms of DFF40 concurrent with caspase-3 activation during acute and chronic liver injury in rats

DNA fragmentation factors (DFF) form protein complexes consisting of nuclease DFF40/CAD and inhibitory chaperon DFF45/ICAD. Although activated caspase-3 has been shown to cleave DFF complexes with the release of active DFF40 and DNA fragmentation, the organ-specific mechanisms of DFF turnover during liver injury accompanied by massive apoptosis are unclear. In this study, we investigated hepatic profile of DFF40-immunopositive proteins in two models of liver injury in rats: acute ischemia/reperfusion (I/R) and chronic alcohol administration. We show that DFF40-like proteins occur in intact rat liver mainly as a 52kDa protein. Hepatic I/R-induced caspase-3 activation and a time-dependent accumulation of DFF40-positive protein fragments (40 and 20kDa), most likely via specific caspase-3 cleavage as evidenced by in vitro digestion of intact liver tissue with recombinant caspase-3. In addition, immunoprecipitation with DFF40 followed by Western blot with active caspase-3 antibody revealed the presence of active caspase-3 in DFF40-immunopositive 20kDa proteins. Chronic alcohol administration in rats also resulted in a dose-dependent fragmentation of DFF40 proteins similar to I/R injury. Collectively, these data demonstrate that DFF40 immunopositive proteins exist in the liver as distinct, tissue-specific molecular forms that may be processed by caspase-3 during both acute and chronic liver injury.     

Roles of DNA fragmentation factor and poly(ADP-ribose) polymerase-1 in sensitization of fibroblasts to tumor necrosis factor-induced apoptosis.

DNA fragmentation factor (DFF) comprises DFF45 and DFF40 subunits, the former of which acts as an inhibitor of the latter (the catalytic subunit) and whose cleavage by caspase-3 results in DFF activation. Disruption of the DFF45 gene blocks the generation of 50-kb DNA fragments and confers resistance to apoptosis. We recently suggested that the early fragmentation of DNA by DFF and the consequent activation of poly(ADP-ribose) polymerase-1 (PARP-1), mitochondrial dysfunction, and activation of caspase-3 contribute to an amplification loop in the apoptotic process. To verify the existence of such a loop, we have now examined the effects of restoring DFF expression in DFF45-deficient fibroblasts. Co-transfection of mouse DFF45(-/-) fibroblasts with plasmids encoding human DFF40 and DFF45 reversed the apoptosis resistance normally observed in these cells. The DFF45(-/-) cells regained the ability to fragment their DNA into 50-kb pieces in response to TNF, which resulted in a marked activation of PARP-1 and a concomitant depletion of intracellular NAD. DFF expression also resulted in an increase both in cytochrome c release into the cytosol and in caspase-3 activation triggered by TNF. These results support the importance of DFF, PARP-1, mitochondria, and caspase-3 in an amplification phase of TNF-induced apoptosis.     

Differential localization of ICAD-L and ICAD-S in cells due to removal of a C-terminal NLS from ICAD-L by alternative splicing

CAD/CPAN/DFF40 is an apoptotic nuclease that is associated with the regulatory subunit ICAD/DFF in healthy cells. ICAD has two forms, ICAD-L/DFF45 and ICAD-S/DFF35, which are transcribed from a single gene by alternative splicing. They differ at the C-terminus: 70 amino acids of ICAD-L are replaced by 4 different amino acids in ICAD-S. We previously showed that both transfected and endogenous ICAD-L are nuclear; however, the localization of ICAD and CAD remains controversial and an important issue to clarify. Here we present the evidence that ICAD-L is nuclear due to the presence of an autonomous nuclear localization signal located in the C-terminal 20 amino acids. This NLS is missing from ICAD-S, which is distributed throughout the cell. We also showed that a GFP:CAD fusion protein is located in the nucleus of transfected cells.     

Subunit structures and stoichiometries of human DNA fragmentation factor proteins before and after induction of apoptosis

DNA fragmentation factor (DFF) is one of the major endonucleases responsible for internucleosomal DNA cleavage during apoptosis. Understanding the regulatory checkpoints involved in safeguarding non-apoptotic cells against accidental activation of this nuclease is as important as elucidating its activation mechanisms during apoptosis. Here we address these issues by determining DFF native subunit structures and stoichiometries in human cells before and after induction of apoptosis using the technique of native pore-exclusion limit electrophoresis in combination with Western analyses. For comparison, we employed similar techniques with recombinant proteins in conjunction with atomic force microscopy. Before induction of apoptosis, the expression of DFF subunits varied widely among the cell types studied, and the chaperone/inhibitor subunits DFF45 and DFF35 unexpectedly existed primarily as monomers in vast excess of the latent nuclease subunit, DFF40, which was stoichiometrically associated with DFF45 to form heterodimers. DFF35 was exclusively cytoplasmic as a monomer. Nuclease activation upon caspase-3 cleavage of DFF45/DFF35 was accompanied by DFF40 homo-oligomer formation, with a tetramer being the smallest unit. Interestingly, intact DFF45 can inhibit nuclease activity by associating with these homo-oligomers without mediating their disassembly. We conclude that DFF nuclease is regulated by multiple pre- and post-activation fail-safe steps.