重组人融合基因Nm23-H1/HbFGF的构建及其在大肠杆菌中表达的研究

根据Nm23-H1与HbFGF cDNA序列,人工合成一段中间核酸序列,将它分别与Nm23-H1 cDNA的上游引物及HbFGF cDNA的下游引物组成两对引物,通过PCR(聚合酶链式反应)构建出融合基因Nm23-H1/HbFGF,将其定向克隆于质粒载体pBV220上,经诱导,SDS-PAGE分析,表达产物分子量为34kD,表达量占菌体总蛋白的14%,表达产物以包涵体形式存在,ELISA和Western印迹表明包涵体具有Nm23-H1和HbFGF抗原性,然后对包涵体进行变性、复性及纯化处理,取纯化产物进行定性和生物活性分析,结果证明纯化产物具有Nm23-H1和HbFGF的抗原性及生物活性。以上实验为今后进一步研究融合基因Nm23-H1/HbFGF在真核细胞中的抑癌、致癌性质打下了基础。     

Nm23-H1/NDPK-A基因在大肠杆菌中的高效表达及产物纯化的研究

利用聚合酶链反应（ＰＣＲ）技术扩增人二磷酸核苷激酶Ａ亚基（ＮＤＰＫ－Ａ）基因,即ｎｍ２３－Ｈ１／ＮＤＰＫ－Ｋ基因的编码序列,经序列分析后,定向克隆于表达质粒载体ｐＢＶ２２０,在大肠杆菌ＤＨ５α中高效表达出重组人ＮＤＰＫ－Ａ．表达产物为可溶性的非融合蛋白,占菌体总蛋白４２％．斑点ＥＬＩＳＡ法鉴定表明表达产物与ＮＤＰＫ－Ａ标准抗血清呈阳性反应．以ＤＥＡＥ纤维素弱阴离子交换层析、ＣｉｂａｃｒｏｎＢｌｕｅ染料亲和层析结合高效液相排阻色谱技术纯化ｒＮＤＰＫ－Ａ,得纯度为９６．７％的目标蛋白．以反相高效液相色谱法进行酶活性分析,表明纯化的ｒＮＤＰＫ－Ａ能催化ＡＴＰ＋ＵＤＰ＝ＡＤＰ＋ＵＴＰ的反应,比活性为８００Ｕ／ｍｇ蛋白．     

抑癌基因nm23—H1在逆转录病毒载体的表达

癌症转移是癌症病人死亡的主要原因之一。抑制肿瘤转移的基因ｎｍ２３－Ｈ１与ｎｍ２３－Ｈ２分别编码二磷酸核苷激酶（ＮＤＰＫ）的Ａ与Ｂ亚基〔１〕,ｎｍ２３－Ｈ１基因能有效地抑制肿瘤转移〔２〕。ｎｍ２３－Ｈ１的表达水平与黑色素瘤〔２,３〕、乳腺癌〔４〕、卵巢...     

重组BPI23—Fcγ1融合蛋白在CHO细胞中的表达

The fusion gene of BPI23 and human Fc gamma 1 was obtained by PCR method, and the expression plasmid was constructed to express recombinant BPI23-Fc gamma 1 fusion protein in CHO cells. After transfection with the plasmid and selection by methotrexate, the cell lines expressing the fusion protein were obtained. The recombinant protein was purified using cation-exchange chromatography and its bioactivity was proved with bactericidal assays.     

人肿瘤坏死因子可溶性受体I与人IgG：Fc的融合基因的构建及其在大？…

采用ＰＣＲ方法引入编码氨基酸残基序列为ＧＧＧＧＳＧＧＧＧＳ的镜头,将ｓＴＮＦＲ１ ｃＤＮＡ与人ＩｇＧ：Ｆｃ ｃＤＮＡ片段连接,构成融合基因ｆｕｓｉｏｎ １。将ｆｕｓｉｏｎ １克隆在ｐＢＳⅡ ＳＫ＾＋载体上。测定序列后,转入ｐＲＳＥＴ－Ｂ表达载体,在大肠杆菌中表达,得到分子量为４５ｋＤａ的蛋白,超声破碎后电泳证明表达的蛋白为包涵体,经免疫蛋白印迹证实为我们构建的融合蛋白。     

Epstein—Barr病毒BNLF—1基因的克隆及其在PA317细胞中？…

本文报道以人Ｅｐｓｔｅｉｎ－Ｂａｒｒ病毒的潜代膜蛋白基因ＢＮＬＦ－１作为目标基因,插入至逆转录病毒载体ｐＺｉｐＮｅｏＳＶ（ｘ）的克隆位点上,由此获得重组逆转录病毒载体ｐＺｉｐＮｅｏＳＶ（ｘ）－ＬＭＰ及其反义载体ｐＺｉｐＮｅｏＳＶ（ｘ）－ａｎｔｉ－ＬＭＰ,通过质粒ＤＮＡ的电转染和Ｇ４１８培养基筛选,然后对１０个抗性克隆进行基因整合分析、获得６个含ＭＬＶ－ＬＴＲ／ＢＶＬＦ－１融合基因的阳性克隆,采用Ｎ     

表达大肠杆菌K88ac-ST1-LTB融合蛋白基因工程菌株的构建

利用PCR技术,从大肠杆菌C83902质粒中扩增出K88ac基因、ST1突变基因和LTB基因,通过分离、纯化、内切酶酶切、连接和转化,构建了含K88ac-ST1-LTB融合基因表达载体的重组菌株BL21（DE3）（pXKST3LT5)。经酶切鉴定和DNA序列分析证实,构建的重组质粒pXKST3LT5中含有K88ac-ST1-LTB融合基因,且基因序列和阅读框架均正确。经ELISA检测,重组菌株表达的K88ac-ST1-LTB融合蛋白能够被ST1单抗、LTB和K88ac抗体识别。经乳鼠灌胃试验证实,表达的融合蛋白已丧失天然ST1肠毒素的活性。免疫实验结果表明,K88ac-ST1-LTB融合蛋白能够诱发小白鼠产生抗体,该抗体具有中和天然ST1肠毒素的毒性作用,表明构建的重组菌株可以作为预防仔猪黄、白痢基因工程菌苗的候选菌株。     

中国人结肠癌nm23H1基因遗传不稳定性的研究

Techniques such as DNA extraction from paraffin-embedded tissues, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), ordinary silver stain, Envision immunohistochemistry and Leica-Qwin computer imaging techniques were used to study microsatellite instability (MSI) and loss of heterozygosity (LOH) of locus D17S396 at the 17th chromosome of Chinese patients and their influence on the expression of gene nm23H1, and to clarify the relationship between the genetic instability of gene nm23H1 and the development of colon cancer, which may provide experimental basis for clinical treatment. In our experiments, the frequency of MSI, LOH and nm23H1 protein reacted positive of 30 cases of colon cancer were 26.67%, 20.00% and 53.33% respectively. In tumor node metastasis (TNM) staging, the positive frequency of MSI (43.75%) and nm23H1 protein (81.25%) in stage I + II were more than those (MSI 7.14%, p < 0.05 and nm23H1 21.43%, p < 0.01) in stage III + IV, while the frequency of LOH (35.71%), which had a rising trend along with the Duke's staging increasing, was higher than that of LOH (6.25%, p < 0.05) in stage I + II. The positive frequency of nm23H1 protein in the group of tubular adenocarcinoma (60.00%) was distinctively higher than that in the group of mucoid adenocarcinoma (20.00%, p < 0.01), showing a rising trend along with the increase of the differentiation degree of tubular adenocarcinoma. Furthermore, the positive frequency of nm23H1 protein in MSI positive group was also higher than MSI negative group (p < 0.05). And there was no difference in nm23H1 protein expression analyzed by computer imaging techniques. The results of experiments indicated that both MSI and LOH controlled the development of sporadic colon cancer independently in different paths. LOH occurred mostly in the late period of sporadic colon cancer and endowed with it a high aggressive and poor prognosis. In contrast, MSI was an early period molecule marker of sporadic colon cancer. Increasing the amount of nm23H1 protein expression could effectively restrain colon cancer metastasis and improved prognosis of sporadic colon cancer patients.     

FMR—1同源基因在大鼠脑发育过程中持续表达

以克隆的人ＦＭＲ－１ ｃＤＮＡ片段为探针，进行ＲＮＡ印迹杂交，检测发育过程中大鼠脑组织ＦＭＲ－１同源基因的表达，结果显示从胚胎早期至出生后一个月该基因有持续表达，其中在胚胎发育晚期表达量较高，提示ＦＭＲ－１基因可能参与胎脑发育的调节。     

Double mutant P96S/S120G of Nm23-H1 abrogates its NDPK activity and motility-suppressive ability

The Nm23-H1 gene is a metastasis suppressor gene. However, its biochemical mechanism of suppressing the metastatic potential of cancer cells is still unknown. The previous hypothesis that a histidine protein kinase activity may contributes to the motility-suppressive effect of Nm23-H1 could not explain why the H118F mutant, a kinase-deficient mutant, still had motility-suppressive ability. We conducted a study on the double mutant P96S/S120G of Nm23-H1 and succeeded in introducing the RP-HPLC method in NDPK assay. The results showed that the double mutant P96S/S120G, when expressed in the bacteria, was completely aggregated in inclusion bodies; this mutant abrogated not only its motility-suppressive ability, but also its NDPK activity. Based on previous work and this study, we prompted that the deficiency of motility-suppressive function of S120G, P96S, and P96S/S120G mutants was due to their altered structure, which might deprive Nm23-H1 of most activities including kinase activity or interactions with other proteins.     

siRNA分析nm23-H1基因与人慢性髓性白血病的关系

设计并筛选靶向nm23-H1基因的siRNAs序列,探讨nm23-H1基因与人慢性髓性白血病之间的关系。依据siRNA设计原则,设计3条siRNA序列。将不同靶点的siRNA用lipofectamine2000转染人慢性髓性白血病细胞株K562。转染后24h RTPCR检测nm23-H1mRNA水平变化;转染后48h免疫细胞化学法检测nm23-H1蛋白表达。MTT法检测转染后24h、48h和72h有效siRNA对K562细胞生长的影响。3条siRNA中,siNM526能有效地抑制K562细胞nm23-H1基因表达,转染siNM526的K56细胞生长受到抑制。说明下调nm23-H1基因的表达有抑制K562细胞增殖的作用,即降低了K562细胞的恶性程度。nm23-H基因有可能成为白血病治疗潜在的分子靶点。     

Nm23-H1 is responsible for SUMO-2-involved DNA synthesis induction after X-ray irradiation in human cells

Human cells derived from nevoid basal carcinoma syndrome (NBCCS) patients show increased levels of DNA synthesis activity after X-ray irradiation which is suggested to be casually related to reduction in cellular amounts of small ubiquitin-like protein modifier (SUMO-2/SMT-3A). In the present study, an increased level of DNA synthesis activity was found 8 h after X-ray irradiation in HeLa cells with reduction in SUMO-2 amounts by siRNA treatment for SUMO-2. When comparative proteomic analysis was performed between the siRNA and mimic control siRNA treated cells using two-dimensional (2D) electrophoresis and mass spectrometry, three proteins were identified as candidates. Our research focused on Nm23-H1, a nucleoside diphosphate kinase, whose amounts decreased after X-ray irradiation in HeLa cells treated with siRNA for SUMO-2. In the Nm23-H1 siRNA treated cells, induction of DNA synthesis was also detected. Furthermore, in synchronized HeLa cells, DNA synthesis was confirmed in the S phase. Moreover, increased expression of proliferating cell nuclear antigen (PCNA) was observed in Nm23-H1 siRNA treated HeLa cells after X-ray irradiation. In addition, Nm23-H1 was modified with SUMO-2 after X-ray irradiation. The present findings suggest that the reduction of Nm23-H1 is related to the decrease in sumoylation, which in turn, is involved in the induction of DNA synthesis via the regulation of PCNA expression after X-ray irradiation.     

Point mutations affecting the oligomeric structure of Nm23-H1 abrogates its inhibitory activity on colonization and invasion of prostate cancer cells

In order to identify Nm23-H1's structural motifs influencing its metastasis-inhibitory activity, we transfected DU 145 human prostate carcinoma cells with the expression vector encoding the Nm23-H1 protein with mutations at the following amino acids: serine-44, a phosphorylation site; proline-96, a site corresponding to the k-pn mutation that causes developmental defects in Drosophila; and serine-120, a site of mutation in human neuroblastoma and phosphorylation. Significant decrease in colonization in soft agar and invasiveness of DU 145 cells was observed in the wild type nm23-H1 transfectants, and also in the serine-44 and serine-120 to alanine mutant nm23-H1-transfected cell lines. However, the k-pn type proline-96 to serine (P96S) and neuroblastoma type serine-120 to glycine (S120G) mutations of Nm23-H1 abrogated its inhibitory activity on colonization and invasion. Meanwhile, all of the recombinant mutant Nm23-H1 proteins produced in Escherichia coli exhibited NDP kinase activity levels at the wild type protein, although the P96S and S120G mutant proteins exhibited decreased histidine protein kinase activity and autophosphorylation level, respectively. Interestingly, only two of the mutant recombinant Nm23-H1 proteins examined, P96S and S120G, exhibited reduced hexameric and increased dimeric oligomerization relative to the wild type. These correlative data suggest that the metastasis-suppressing activity of Nm23-H1 may depend on its oligomeric structure, but not on its NDP kinase activity.     

Ad-GFP-nm23-H1裸鼠体内抑制人恶性黑色素瘤A375作用的研究

目的探讨Ad—GFP—nm23-H1对人恶性黑素瘤裸鼠皮下移植瘤的抑制作用,从而为后期nm23-H1基因和腺病毒载体用于人恶性黑色素瘤及其它肿瘤的基因治疗提供一定的理论和方法。方法在裸鼠真皮下建立人A375细胞黑色素瘤动物模型后,设对照组及10^9 PFU／ml、10^10 PFU／ml的Ad—GFP—nm23-H1干预组。经Ad—GFP—nm23-H1干预治疗后,取瘤体称重,计算抑瘤率,通过光镜进行瘤组织病理形态学观察。结果对照组及10^9 PFU／ml、10^10 PFU／ml的Ad—GFP-nm23-H1干预组的平均肿瘤体积和瘤重分别为：1．4129&#177;0．4832mm^3、1．1914&#177;0．3304mm^3、0．75&#177;0．2548mm^3和1．924&#177;0．539g、1．655&#177;0．5754g、1．195&#177;0．2639g。与另两组比较,10^10 PFU／ml的Ad．GFP—nm23-H1干预组对A375具有明显的抑制作用。结论10^10 PFU／ml的Ad—GFP—nm23-H1干预组对裸鼠移植A375实体瘤有明显的抑制作用。     

腺病毒介导的nm23-H1对人结直肠癌裸鼠移植作用的实验研究

目的 探讨Ad-GFP-nm23-Hl对人高转移结直肠癌移植瘤的抑制作用,为临床肿瘤基因治疗提供一定的理论依据.方法 将15只人结直肠癌移植瘤裸鼠随机分为高剂量组(1010 PFU/ml Ad-GFP-nm23-Hl)、低剂量组(109 PFU/ml Ad-GFP-nm23-Hl)、阴性对照组(PBS).均采用瘤体注射给药,连续给药4 d,每2 d测一次肿瘤体积.观察10 d后取瘤块称重和组织病理学检查.结果 Ad-GFP-nm23-Hl高剂量组移植瘤体积较其他各组明显缩小(P<0.05),瘤体积抑制率和瘤重抑制率分别为76%和66%. 结论 Ad-GFP-nm23-Hl对人高转移结直肠癌移植瘤有抑制作用,并存在剂量依赖性.     

nm23-H1基因缺失人肺癌细胞株的筛选与鉴定

nm23一Hj基因与肺癌的侵袭与转移密切相关,但是其作用的分子机制尚不清楚,为研究nm23—141基因的功能,筛选并鉴定了nm23一H,基因缺失人肺癌细胞株及其生物学特性．应用SoutheITIblot．RT—PCR和West—elTl blot检测9株人肺癌细胞株中nm23—14,基因的存在状态及其生物学行为．结果发现发现人大肺癌细胞株L9981中存在nm23—141等位基因的杂合性缺失,与其同源的NL9980及其它7株肺癌细胞株中nm23—111基因均以杂合子的形式存在;并且L998l细胞株的增殖能力、克隆形成能力、体外侵袭力．裸鼠体内成瘤性及移植瘤肺转移的能力均显著高于NL9980．研究结果显示nm23一H,基因的缺失可能与L998l细胞株恶性表型和高转移潜能密切相关．     

nm23-H1基因转染L9981肺癌细胞前后基因表达谱的变化

应用基因芯片检测L9981细胞转染nm23-H1基因前后细胞基因表达谱的改变.提取L9981细胞转染nm23-H1基因前后细胞的总RNA,纯化为mRNA后再转录为cDNA.cDNA经限制性内切酶Sau3AI消化后,cDNA片段分别用cy3和cy5标记,与定制的包含14000个基因芯片杂交.杂交结果经扫描和软件分析,nm23-H1基因转染L9981细胞后发现1156(8.26%,1156/14000)个基因表达上调,而642(4.59%,642/14000)个基因表达下调.涉及基因包括信号传导、癌基因与抑癌基因、转移相关基因、细胞周期与凋亡、细胞外基质与细胞骨架相关基因,以及细胞因子和转录因子等.nm23-H1基因是通过对转移相关基因的调节来发挥其抑制肺癌细胞株L9981侵袭和转移作用的.