人类博卡病毒(HBoV)非结构蛋白NS1基因的克隆、原核表达及多克隆抗体的制备

目的:克隆、表达、纯化人类博卡病毒(HBoV)非结构蛋白NS1,制备抗NS1多克隆抗体。方法:利用PCR扩增HBoV非结构蛋白NS1基因,将其克隆至pMAL-c2X表达载体上,重组质粒转化大肠杆菌DH10B,IPTG诱导表达。表达的融合蛋白经Amylose Resin亲和层析柱纯化后,免疫新西兰大白兔制备多克隆抗体。用间接ELISA法检测抗体效价。结果:原核表达融合蛋白MBP-NS1,并获得了其多克隆抗体,抗体效价达到1∶32000。结论:在原核表达系统中表达、纯化了融合蛋白,制备抗NS1多克隆抗体,为进一步研究该病毒非结构蛋白基因的转录和翻译机制提供可靠的工具。     

神经纤毛蛋白-1的原核表达及兔抗小鼠神经纤毛蛋白-1抗体免疫学活性的研究

目的:在原核系统中分段表达神经纤毛蛋白-1(Nrp1);将纯化的蛋白免疫家兔后获得特异的抗体,并将其应用于检测组织和细胞中Nrp1分子的表达。方法:提取BALB/c胎鼠脑组织总RNA,通过RT-PCR分段扩增获得Nrp1基因片段,将PCR产物插入表达载体pET28a( ),获得含5个Nrp1基因片段的重组质粒,在大肠杆菌BL21(DE3)中诱导蛋白表达并纯化;将纯化的重组蛋白免疫新西兰大白兔获得针对目的蛋白的特异性多克隆抗体;利用Nrp1特异性多抗检测胎鼠脑组织和HeLa细胞中Nrp1的表达。结果:在原核系统中分段表达了Nrp1蛋白,通过Ni-NTA纯化了Nrp1蛋白片段;纯化的Nrp1蛋白免疫新西兰大白兔获得了具有免疫活性的多抗;兔抗小鼠Nrp1特异性多抗可用于检测组织、真核细胞中Nrp1的表达。结论:应用原核系统成功地表达了Nrp1蛋白,兔抗小鼠Nrp1特异性多抗可用于免疫学检测Nrp1分子的表达。     

绵羊精子赤道膜蛋白片段1(SPESP1)的克隆、原核表达及纯化

旨在克隆绵羊Spesp1 c DNA并表达,得到纯化的GST-SPESP1融合蛋白。以绵羊睾丸组织总c DNA为模板,根据Gen Bank公布的绵羊基因组序列设计引物,PCR扩增得到Spesp1 c DNA,构建原核表达重组载体p GEX-Spesp1,将其转化到E.coliBL21中表达,并优化其表达条件,利用SDS-PAGE切胶纯化法得到纯化的融合蛋白,用Western blot鉴定所得融合蛋白。结果表明,经测序,克隆得到的Spesp1 c DNA序列与Gen Bank中预测的c DNA序列对比有两个碱基不同,并造成一个氨基酸的差异。在E.coliBL21中成功表达重组融合蛋白,其最优表达条件为:37℃、4 h、0.005%IPTG终浓度,SDS-PAGE和Western blotting中,融合蛋白位于约64 k D处并可以被抗GST和抗羊SPESP1的抗体识别,与预计相符,表明融合蛋白成功表达。成功纯化得到GST-SPESP1融合蛋白,为研究SPESP1在精卵细胞膜融合中的功能奠定了基础。     

风疹病毒(RV)包膜糖蛋白E1特异基因片段的原核表达

目的:表达风疹病毒(RV)E1特异肽段的重组融合蛋白.方法:经过双酶切鉴定和测序鉴定的阳,陛重组质粒载体pGEX-2T/E1-N,转化到感受态大肠杆菌BL21后,用异丙基-β-D硫代半乳糖苷(IPTG)诱导其表达,并对诱导条件进行优化.用谷胱甘肽琼脂糖珠纯化重组融合蛋白,SDS-PAGE鉴定.结果:用IPTG可以诱导E1特异肽段的重组融合蛋白表达,37℃诱导时,最佳诱导剂浓度为1 mmol/L,最佳诱导时间为4h.诱导温度从37℃降至16℃时,重组融合蛋白以可溶性形式表达,用谷胱甘肽琼脂糖珠纯化获得了纯化的重组融合蛋白.结论:利用原核表达系统可以获得纯化的风疹病毒E1特异肽段的重组融合蛋白.     

HIV-1逆转录酶的表达、纯化及晶体生长条件的摸索

利用大肠杆菌原核表达系统对HIV-1 RT蛋白进行表达,纯化以及晶体生长条件的搜索。以PNL4-3为模板,通过聚合酶链式反应扩增出HIV-1 RT蛋白的基因,将扩增出的目的基因克隆到表达载体Pet-duet中,构建HIV-1逆转录酶的重组质粒,重组质粒转化表达菌株BL21(DE3)进行诱导表达。HIV-1逆转录酶主要以可溶形式表达,经亲和柱Ni2+-NTA,阴离子交换柱MONOQ,分子筛进行纯化后获得了蛋白,其纯度达到85%-90%,基本满足了晶体生长条件的要求。对晶体生长条件行了初筛,最终并获得了HIV-1 RT的蛋白晶体,为蛋白晶体的优化以及蛋白结构的研究奠定了基础。     

人热休克蛋白转录因子1 DNA结合结构域的表达、纯化和晶体生长

目的:获得大量热休克转录因子1(HSF1)DNA结合结构域(DBD)蛋白,用于晶体生长的三维结构解析。方法:将DBD基因片段克隆至原核表达载体pGEX-6P-1中并获得高效表达,经过Glutathione SepharoseTM 4B亲和层析、ResourceQ纯化后,蛋白纯度达到95%以上。结果:圆二色谱仪分析蛋白质的二级结构结果显示α螺旋占33%,β折叠占15%;采用悬滴气相扩散法得到了针状DBD晶体。结论:纯化的蛋白质与同源性达68%的Kluyveromyceslactis的DBD有相似的空间构象。获得的蛋白质晶体为进一步的三维结构解析奠定了基础。     

人源Plk1及其结构域蛋白的融合表达及纯化

目的:表达纯化GST-Plk1及GST-Plk1 KD、GST-Plk1 PBD融合蛋白,以用于Plk1与其结合蛋白相互作用的研究。方法:PCR扩增Plk1全长及其KD、PBD结构域基因,定向克隆至p GEX-4T-1原核表达载体中,在大肠杆菌中分别表达其融合蛋白,并利用GSH交联的琼脂糖珠纯化。结果:人源Plk1及其结构域基因被克隆至p GEX-4T-1载体中;通过亲和纯化获得带有GST标签的Plk1及其结构域的融合蛋白。结论:构建了GST-Plk1、GST-Plk1 KD、GST-Plk1 PBD表达质粒并表达了相应的融合蛋白,为研究Plk1体外相互作用蛋白提供了基础。     

GST-Ets-1融合蛋白的表达与纯化

[目的]旨在原核表达Ets-1基因,纯化获得GST-Ets-1融合蛋白。[方法]以SD大鼠脑垂体cDNA为模板,利用PCR扩增含有Bam HI和Not I酶切位点的Ets-1基因;然后将其克隆到pGEX-4T-1原核表达载体中,将正确的重组载体转入大肠杆菌BL21(DE3);用IPTG诱导表达,再利用Magne GST particles亲和纯化GST-Ets-1融合蛋白;最后通过Western blot鉴定此融合蛋白。[结果]成功构建pGEX-4T-1-Ets-1原核表达载体;30℃条件下,0.2 mmol/L的IPTG能诱导出大量的可溶性GST-Ets-1蛋白;经Magne GST particles纯化的GST-Ets-1蛋白可被识别ETS-1的抗体特异识别。[结论]纯化的GST-Ets-1蛋白可用于后续的生物学研究。     

中华蜜蜂SRP9基因的克隆、序列分析及原核表达

【目的】探究中华蜜蜂Apis cerana cerana信号识别颗粒9(signal recognition particle 9,SRP9)基因的序列,获得纯化的重组蛋白,为探究其蛋白功能奠定基础。【方法】利用RT-PCR方法,以中华蜜蜂的c DNA为模板扩增中华蜜蜂SRP9基因编码区,并通过软件MEGA5.1构建系统发育树,运用Predice Protein和SWISS-MODEL等软件预测分析蛋白结构;用大肠杆菌Escherichia coli表达系统进行原核表达;采用尿素洗脱法进行重组蛋白纯化。【结果】扩增得到的中华蜜蜂SRP9基因编码区长为237 bp,命名为Acc SRP9。该基因编码78个氨基酸,编码蛋白的相对分子量为9.36 k D,等电点为9.17。系统进化树分析表明,Acc SRP9与西方蜜蜂Apis mellifera的SRP9和小蜜蜂Apis florea的SRP9聚成一支。蛋白质二级结构预测发现其含有2个α-螺旋区和3个β-折叠区。利用同源建模获得蛋白质的三维结构。将Acc SRP9连入表达载体p GEX-6P-1并在BL21(DE3)中进行原核表达,发现该重组蛋白表达在包涵体中,经蛋白纯化,最终获得了N端带GST标签的重组蛋白p GEX-6P-1-SRP9。【结论】本研究成功克隆了中华蜜蜂SRP9基因Acc SRP9,对其序列进行了分析,并获得了带有标签的重组蛋白,为进一步研究该基因的功能提供参考和材料。     

人纤溶酶原饼环区5(hPK5)基因的分泌型表达

构建人纤溶酶原饼环区5(hPK5)基因的原核可溶性表达载体并进行表达和纯化,获取大量高纯度、具有生物活性的hPK5蛋白。以纤溶酶原cDNA为模板,PCR扩增了hPK5基因,经过适当酶切后构建表达载体pET22b(+)hPK5,转入大肠杆菌BL21(DE3)进行表达并经组氨酸亲和层析获得纯化。带有重组质粒pET22b(+)hPK5的大肠杆菌经IPTG诱导后以可溶性形式表达16kDa的蛋白,其表达量占菌体总蛋白的30%以上,纯化后目的蛋白纯度达95%以上,Western印迹表明重组蛋白具有Histag抗原活性。构建了pET22b(+)hPK5重组质粒并成功地在大肠杆菌中获得可溶性表达,为获得大量hPK5基因工程产品奠定了实验基础。     

ZFP36L1通过下调细胞周期蛋白D1抑制舌癌细胞增殖

C3H1型的锌指蛋白36 (zinc finger protein 36,C3H type-like 1,ZFP36L1)是一种高度保守且具有CCCH型RNA结合结构域的蛋白质。近年来,ZFP36L1在多种肿瘤中的作用被报道,但是在舌癌中的表达型和作用机制尚不清楚。Western印记结合荧光定量PCR检测发现,ZFP36L1在舌癌细胞中的表达明显低于人永生化表皮细胞Hacat。在相对低表达ZFP36L1的舌癌细胞SCC15和SCC25中,稳定过表达ZFP36L1,细胞计数实验发现,SCC15细胞的数目由(4.768±0.09225)×10~3个降低到(3.089±0.09745)×10~3个,SCC25细胞的数目由(6.274±0.01311)×10~3个降低到(4.037±0.01173)×10~3个;平板克隆实验提示,SCC15和SCC25细胞克隆数目是对照组的0.67倍,0.68倍,0.7倍和0.59倍,0.57倍,0.59倍;过表达ZFP36L1组G_1期的SCC15和SCC25细胞分别由61.82±0.8933%增加到88.72%±0.8378,由56.31%±1.029增加到71.7%±0.9303;而S期的细胞由25.21%±0.9865减少到11.31%±0.6567,由28.58%±0.8182减少到18.61%±0.6798。过表达ZFP36L1能明显下调SCC15和SCC25细胞中细胞周期蛋白D1(cyclinD1)的蛋白质水平。过表达ZFP36L1组的SCC15和SCC25细胞中,细胞周期蛋白D1 mRNA的表达量分别是对照组的0.217倍和0.175倍。在舌癌细胞中,上调细胞周期蛋白D1的表达水平可消除由过表达ZFP36L1引起的细胞增殖能力降低。总之,ZFP36L1在舌癌中呈低表达;可通过下调细胞周期蛋白D1的表达,抑制舌癌细胞增殖。     

人IGF-1在大肠杆菌中的可溶表达和纯化

目的:在大肠杆菌中的可溶表达和纯化人胰岛素样生长因子1(hIGF-1)。方法:根据hIGF-1的氨基酸序列和大肠杆菌密码子偏爱性,利用重叠延伸PCR的方法合成hIGF-1DNA序列,构建表达载体,在大肠杆菌OrigamiB(DE3)中与硫氧还蛋白TrxA融合表达,并通过盐析和镍柱亲合层析进行纯化。结果:SDS-PAGE分析显示,重组融合蛋白以可溶形式存在,分子量约为28kDa,占上清总蛋白的50%以上。经盐析和镍柱亲合层析进行纯化,目标蛋白纯度可达到90%左右。结论:复合干扰素在大肠杆菌中的高效可溶表达。     

Cytostatic effect of inostamycin, an inhibitor of cytidine 5'-diphosphate 1,2-diacyl-sn-glycerol (CDP-DG): inositol transferase, on oral squamous cell carcinoma cell lines.

Inostamycin, which was recently isolated from Streptomyces sp. MH816-AF15 as an inhibitor of cytidine 5'-diphosphate 1,2-diacyl-sn-glycerol (CDP-DG): inositol transferase, caused a G1-phase accumulation in the cell cycle of small cell lung carcinomas. To investigate whether the cytostatic effect of inostamycin is restricted to lung carcinoma cell lines or applicable to other type of cells, we tested five oral squamous cell carcinoma (SCC) cell lines. Cell growth was suppressed in 62.5--125 ng/ml inostamycin in the culture medium in all oral cancer cell lines tested, with non-viable cells being <1%, indicating inostamycin is cytostatic on SCC cell lines. Decrease in cyclin D1 mRNA and protein expression due to the inostamycin treatment was accompanied by suppression of phosphorylated retinoblastoma susceptibility gene product (pRB-P) levels. Moreover, flow cytometric analysis showed that inostamycin induced an increase in G1/G0 cells (1.2--3.2 fold) over 24 h. These results suggest that inostamycin is a useful agent for tumour dormant cytostatic therapy for oral SCC.     

Amphiregulin is a vitamin D3 target gene in squamous cell and breast carcinoma

1alpha,25-Dihydroxyvitamin D(3) [1,25(OH)2D3] inhibits growth of cells derived from a variety of tumors in vitro and in vivo. Proliferation in vitro of human SCC25 cells, derived from a primary squamous cell carcinoma (SCC) of the tongue, was blocked by 1,25(OH)2D3 and its analog EB1089. A similar effect was observed with 13-cis retinoic acid (RA), which has been used in chemoprevention of SCC. We identified amphiregulin, a member of the epidermal growth factor family, as a 1,25(OH)2D3 target gene in SCC25 cells. Induction of amphiregulin mRNA by 1,25(OH)2D3 was rapid and sustained over 48 h, and was unaffected by cycloheximide. 1,25(OH)2D3 also induced amphiregulin mRNA in estrogen receptor-positive and -negative human breast cancer cell lines, but not in LNCaP human prostate cancer cells. RAR- or RXR-specific retinoids did not affect amphiregulin mRNA levels in SCC25 cells; however, 13-cis RA partially blocked the response to 1,25(OH)2D3. Amphiregulin partially inhibited growth of SCC25 cells in culture. Our data show that amphiregulin is a 1,25(OH)2D3 target gene, and suggest that its induction may contribute to the growth inhibitory effects of 1,25(OH)2D3.     

MCPIP1 overexpression in human neuroblastoma cell lines causes cell-cycle arrest by G1/S checkpoint block

Monocyte chemoattractant protein-1-induced protein 1 (MCPIP1) has a multidomain structure, which assures its pleiotropic activity. The physiological functions of this protein include repression of inflammatory processes and the prevention of immune disorders. The influence of MCPIP1 on the cell cycle of cancer cells has not been sufficiently elucidated. A previous study by our group reported that overexpression of MCPIP1 affects the cell viability, inhibits the activation of the phosphoinositide-3 kinase/mammalian target of rapamycin signalling pathway, and reduces the stability of the MYCN oncogene in neuroblastoma (NB) cells. Furthermore, a decrease in expression and phosphorylation levels of cyclin-dependent kinase (CDK) 1, which has a key role in the M phase of the cell cycle, was observed. On the basis of these previous results, the purpose of our present study was to elucidate the influence of MCPIP1 on the cell cycle of NB cells. It was confirmed that ectopic overexpression of MCPIP1 in two human NB cell lines, KELLY and BE(2)-C, inhibited cell proliferation. Furthermore, flow cytometric analyses and imaging of the cell cycle with a fluorescence ubiquitination cell-cycle indicator test, demonstrated that overexpression of MCPIP1 causes an accumulation of NB cells in the G1 phase of the cell cycle, while the possibility of an increase in G0 phase due to induction of quiescence or senescence was excluded. Additional assessment of the molecular machinery responsible for the transition between the cell-cycle phases confirmed that MCPIP1 overexpression reduced the expression of cyclins A2, B1, D1, D3, E1, and E2 and decreased the phosphorylation of CDK2 and CDK4, as well as retinoblastoma protein. In conclusion, the present results indicated a relevant impact of overexpression of MCPIP1 on the cell cycle, namely a block of the G1/S cell-cycle checkpoint, resulting in arrest of NB cells in the G1 phase.     

Cyclin D1 downregulation is important for permanent cell cycle exit and initiation of differentiation induced by anchorage‐deprivation in human keratinocytes

To understand the relationship between permanent cell cycle exit and differentiation the immortalized keratinocyte cell line, SIK and the squamous cell carcinoma, SCC9 were compared during differentiation induced by anchorage‐deprivation. The SIK cells when placed in suspension culture promptly lost almost all ability to reinitiate growth by 2 days concomitantly expressing the differentiation specific proteins, transglutaminase (TGK) and involucrin. These cells rapidly underwent G1 cell cycle arrest with complete disappearance of phosphorylated RB. In contrast SCC9 cells neither showed TGK expression nor increase in involucrin. They decreased their colony‐forming ability much more slowly, which coordinated well with a gradual decrease in phosphorylated RB, demonstrating the significant resistance to loss of colony‐forming ability and cell cycle exit. In accordance, cyclin D1, a positive regulator of cyclin‐dependent kinase (CDK) 4/6 which phosphorylates RB decreased drastically in anchorage deprived SIK but not in SCC9 cells. Endogenous cyclin D1 knockdown in SCC9 cells by siRNA enhanced loss of the colony‐forming ability during anchorage‐deprivation. Conversely enforced expression of cyclin D1 in SIK cells and in another immortalized keratinocyte cell line, HaCaT, partly prevented loss of their colony‐forming abilities. Cyclin D1 overexpression antagonized Keratin 10 expression in suspended HaCaT cells. The result demonstrates the importance of cyclin D1 down regulation for proper initiation of keratinocyte differentiation. J. Cell. Biochem. 106: 63–72, 2009. © 2008 Wiley‐Liss, Inc.     

Polo-like kinase 1 (Plk1) in non-melanoma skin cancers

Polo-like kinase 1 (Plk1) is becoming an increasingly attractive target for cancer management. Plk1 has been shown to be over-expressed in a variety of cancers; however its role in skin cancers is not well-understood. We recently demonstrated that Plk1 is over-expressed in human melanoma and gene-knockdown as well as chemical-inhibition of Plk1 resulted in a significant decrease in melanoma cell viability and growth without affecting the growth of the normal human epidermal melanocytes (NHEMs). Further, the observed anti-proliferative response of Plk1 was found to be accompanied with a significant G2/M cell cycle arrest, mitotic catastrophe and induction of apoptosis in melanoma cells. In this study, we determined the expression profile of Plk1 in non-melanoma skin cancers viz. basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). Our data demonstrated that like melanoma, Plk1 is significantly over-expressed in BCC and SCC samples. Further, we also found that compared to normal human epidermal keratinocytes (NHEKs), Plk1 was over-expressed at both the protein and mRNA levels in squamous A253 and A431 cells. In addition, a similar protein expression pattern was found for the downstream targets of Plk1, viz. Cdk1, Cyclin B1 and Cdc25C. We believe that the expression pattern of Plk1 in the various skin cancers, the insusceptibility of normal keratinocytes, to Plk1 inhibition and the easy accessibility for topical applications lends the skin as an attractive tissue for Plk1 based cancer chemoprevention and chemotherapeutic applications.