rNM23-H1/NDPK-A中试纯化工艺研究

为比较3种DEAE填料对rNM23-H1/NDPK-A的纯化效果,利用同一批中试发酵样品在相同的条件下进行离子交换层析,分别收集P0．2和P1．0两个洗脱峰。通过对洗脱峰中蛋白质含量、目标蛋白相对含量以及酶比活的测定,计算得出Matrex Cellufine A-200,DEAE Sephadex A-25,Macro-Prep DEAE Support三种填料相对应的NDPK－A得率及纯化倍数分别为74．5％、40．8％、92．6％、2．4、1．9、3．1倍。综合分析表明Macro-Prep DEAE Support填料对rNM23-H1/NDPK-A的纯化效果最好。     

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．     

表达人胰高血糖素样肽前药工程菌遗传稳定性

通过传代培养的方法,研究高表达重组人胰高血糖素样肽前药(Pro-rhGLPs)的工程菌E.coliBL21(DE3)/pET41a(+)-hGLPs的遗传稳定性,观察菌体和菌落形态,比较在有无选择压力下的质粒稳定性,酶切和测序鉴定重组基因片断的正确性,SDS-PAGE电泳证实重组蛋白质的表达量的稳定性,在C57BL/6小鼠上进行葡萄糖耐量实验检测重组蛋白生物学活性。结果显示:此工程菌连续传代过程中,保持大肠杆菌的典型特征,各代质粒的酶切鉴定和测序正确,重组蛋白表达水平及生物活性也无显著差异。因此,工程菌E.coliBL21(DE3)/pET41a(+)-hGLPs具有良好的遗传稳定性。     

工程菌DH5α/pCW-PL-XE-TNFαm2的遗传稳定性

目的本基因工程大肠杆菌DH5α/pCW-PL-XE-TNFαm2所表达的靶向融合蛋白XE-TNFαm2已被初步证明具有用于清除艾滋病患者体内HIV病毒的前景。其目的蛋白表达水平为32%～36%细胞总蛋白。本研究旨在验证其遗传稳定性。方法工程菌株DH5α/pCW-PL-XE-TNFαm分别在LBAmp＋与LBAmp-二种固体培养基上逐日单菌落划线传代,32℃培养过夜。每间隔十代运用一般温控表达技术,确定其XE-TNFαm2的蛋白含量,最后比较分析各代之间目的蛋白（20.3 kDa）表达水平的差异情况。结果该重组基因工程菌连续传100代后XE-TNFαm2的蛋白表达水平没有明显差异（P〉0.05）;只是在上述两种情况下传至100代后将其置于4℃保藏4、5、6个月,其目的蛋白表达水平有8%的下降。本载体质粒含有的CIts857序列、PL启动子与T1T2末端终止序列,是确保目的基因稳定高表达的3个关键元件。结论本研究结果证明该工程菌DH5α/pCW-PL-XE-TNFαm2具有良好的遗传稳定性。     

表达H5N1亚型禽流感病毒HA基因重组腺病毒的遗传稳定性分析及滴度测定

研究表达H5N1亚型禽流感病毒HA基因重组腺病毒pAd-H5的遗传稳定性及重组病毒的滴度测定。将重组腺病毒pAd-H5在293细胞上连续传代20次,取第5、10、15和20代的重组病毒采用PCR 方法扩增禽流感病毒HA基因,并进行基因序列测定分析;用标记为GFP的快速测定法计算出20代次时重组病毒的滴度。从各代重组病毒DNA 中均扩增出了约1 700bp的目的条带,与HA基因片段长度一致,基因序列分析表明：第5 、10 、15 代重组病毒中的HA基因序列与原始转移载体序列完全一致,第20代重组病毒插入基因有1处发生了点突变(即HA基因417位A→G),但其编码的氨基酸未发生变化(即同义突变),表明表达的目的蛋白抗原表位未发生变化;计算出的重组病毒滴度为108.875pfu/0.1ml。重组腺病毒pAd-H5在293细胞上连续传代20次,具有良好的遗传稳定性,重组腺病毒的病毒滴度相对较高。     

产harpin的成团泛菌工程菌308R（pCPP430）遗传稳定性的研究

成团泛菌工程菌３０８Ｒ（ｐＣＰＰ４３０）带有梨火疫欧文氏杆菌的与过敏反应和致病性有关的基因簇 （ｈｒｐ）,可以产生能诱导植物抗病性的蛋白质ｈａｒｐｉｎ。该工程菌在ＬＢ液体培养基中生长５０代后,带有重 组质粒ｐＣＰＰ４３０的细胞占总菌量的 １％,带有载体ｐＣＰＰ９的细胞为４６％。工程菌喷雾到番茄叶面,保湿条 件下叶面菌量维持在 １０５ｃｆｕ／ｃｍ２以上,其中带有 ｐＣＰＰ４３０质粒的菌维持在 ４０％以上,带有 ｐＣＰＰ９载体的 菌维持在８０％以上。因此,携带ｈｒｐ基因簇的质粒ｐＣＰＰ４３０在宿主菌中是不稳定的。文中讨论了改进工 程菌遗传稳定性的途径。     

人干扰素α1b突变体IFNα1b/31K的构建及其生物学评价

目的:构建干扰素α1b突变体IFNα1b/31K,以期获得高效低毒的新型药物分子。方法:根据合理药物设计,采用定点突变技术,将干扰素α1b第31位氨基酸残基突变为K,并构建表达IFNα1b/31K重组蛋白。纯化后,对其抗病毒活性、抗肿瘤细胞增殖活性和动物体内急性毒性进行考察。结果:IFNα1b/31K表达量占菌体总蛋白的30%以上。纯化后的IFNα1b/31K纯度大于95%,比活性约为IFNα1b的1.7倍,抗肿瘤增殖活性比IFNα1b降低,未见对实验动物的急性毒性作用。结论:成功设计构建并表达了高效低毒的IFNα1b突变蛋白分子。     

重组痘苗狂犬病毒糖蛋白的构建、表达及其遗传稳定性研究

狂犬病毒糖蛋白基因的痘苗病毒表达质粒 pWS 4在鸡胚细胞内与野生型痘苗病毒天坛株 (痘苗病毒 )同源重组 ,经蚀斑纯化 ,获得表达狂犬病毒糖蛋白基因的重组痘苗病毒 (重组病毒 )。该重组病毒DNAdotblot显示强阳性信号 ,间接免疫荧光试验胞膜及胞浆均见到强阳性荧光反应 ,Westernblot分析仅在 6 4ku处呈现一条狂犬病毒非融合性糖蛋白带。免疫小鼠和狗 ,第 2 8d抗狂犬病毒中和抗体滴度分别达 2 4 30和 >6 96。初免后 14d用 5 0～ 10 0LD50 狂犬病毒CVS株对小鼠脑内攻击 ,保护率达 80 %以上。致病性明显减弱。从第 11代起连续传至 30代 ,病毒纯度、病毒滴度、血凝滴度、蛋白的表达、抗原活性和保护性均无差异 ,说明该重组病毒有良好的遗传稳定性。     

产harpin的成团泛菌工程菌308R(pCPP430)遗传稳定性的研究*

成团泛菌工程菌３０８Ｒ（ｐＣＰＰ４３０）带有梨火疫欧文氏杆菌的与过敏反应和致病性有关的基因簇 （ｈｒｐ）,可以产生能诱导植物抗病性的蛋白质ｈａｒｐｉｎ。该工程菌在ＬＢ液体培养基中生长５０代后,带有重 组质粒ｐＣＰＰ４３０的细胞占总菌量的 １％,带有载体ｐＣＰＰ９的细胞为４６％。工程菌喷雾到番茄叶面,保湿条 件下叶面菌量维持在 １０^５ｃｆｕ／ｃｍ２以上,其中带有 ｐＣＰＰ４３０质粒的菌维持在 ４０％以上,带有 ｐＣＰＰ９载体的 菌维持在８０％以上。因此,携带ｈｒｐ基因簇的质粒ｐＣＰＰ４     

nm23-H1与白血病和淋巴瘤的预后关系

肿瘤转移相关蛋白nm23-H1/NDPK-A在某些癌症中与肿瘤转移潜能呈负相关,另一方面它又是多种血液肿瘤的分化抑制因子。大量临床病例的研究表明nm23-H1在急性髓性白血病和非霍奇金淋巴瘤中具有预后作用。本文主要就nm23-H1/NDPK-A蛋白在急性髓性白血病和非霍奇金淋巴瘤与邓后的相关性以及潜在的预后价值进行了综述。     

Mass spectrometry detection of histidine phosphorylation on NM23-H1

Phosphorylation is a ubiquitous protein post-translational modification that is intimately involved in most aspects of cellular regulation. Currently, most proteomic analyses are performed with phosphorylation searches for serine, threonine, and tyrosine modifications, as the phosphorylated residues of histidine and aspartic acid are acid labile and thus undetectable with most proteomic methodologies. Here, we present a novel buffer system to show histidine phosphorylation of NM23-H1, the product of the first identified putative human metastasis suppressor gene (NME1), which catalyzes the transfer of the γ-phosphate from nucleoside triphosphates to nucleoside diphosphates. On the basis of a pH titration of LC elution buffers and MS/MS identification, recombinant NM23-H1 subjected to autophosphorylation was shown to contain phosphorylated histidine at residue 118 at pH 5 and 6, with each level giving over 75% peptide coverage for identification. The solvent system presented permits the detection of all five possible phosphorylation moieties. Application of histidine and aspartic acid phosphorylation modifications to proteomic analyses will significantly advance the understanding of phosphorylation relay signaling in cellular regulation, including elucidation of the role of NM23-H1 in metastasis.     

The centrosomal kinase Aurora-A/STK15 interacts with a putative tumor suppressor NM23-H1

Alterations in the activity of the centrosomal kinase, Aurora-A/STK15, have been implicated in centrosome amplification, genome instability and cellular transformation. How STK15 participates in all of these processes remains largely mysterious. The activity of STK15 is regulated by phosphorylation and ubiquitin-mediated degradation, and physically interacts with protein phosphatase 1 (PP1) and CDC20. However, the precise roles of these modifications and interactions have yet to be fully appreciated. Here we show that STK15 associates with a putative tumor and metastasis suppressor, NM23-H1. STK15 and NM23 were initially found to interact in yeast in a two-hybrid assay. Association of these proteins in human cells was confirmed by co-immunoprecipitation from cell lysates and biochemical fractionation indicating that STK15 and NM23-H1 are present in a stable, physical complex. Notably, SKT15 and NM23 both localize to centrosomes throughout the cell cycle irrespective of the integrity of the microtubule network in normal human fibroblasts.     

The metastasis suppressor NM23-H1 possesses 3'-5' exonuclease activity

NM23-H1 belongs to a family of eight gene products in humans that have been implicated in cellular differentiation and development, as well as oncogenesis and tumor metastasis. We have defined NM23-H1 biochemically as a 3'-5' exonuclease by virtue of its ability in stoichiometric amounts to excise single nucleotides in a stepwise manner from the 3' terminus of DNA. The activity is dependent upon the presence of Mg(2+), is most pronounced with single-stranded substrates or mismatched bases at the 3' terminus of double-stranded substrates, and is inhibited by both ATP and the incorporation of cordycepin, a 2'-deoxyadenosine analogue, into the 3'-terminal position. The 3'-5' exonuclease activity was assigned to NM23-H1 by virtue of: 1) precise coelution of enzymatic activity with wild-type and mutant forms of NM23-H1 protein during purification by hydroxylapatite and gel filtration column high performance liquid chromatography and 2) significantly diminished activity exhibited by purified recombinant mutant forms of the proteins. Lysine 12 appears to play an important role in the catalytic mechanism, as evidenced by the significant reduction in 3'-5' exonuclease activity resulting from a Lys(12) to glutamine substitution within the protein. 3'-5' Exonucleases are believed to play an important role in DNA repair, a logical candidate function underlying the putative antimetastatic and oncogenic activities of NM23-H1.     

Potential contributions of antimutator activity to the metastasis suppressor function of NM23-H1

nm23-h1 is a well-documented metastasis suppressor gene whose mechanism(s) of action have yet to be fully elucidated. The purpose of this report is to discuss recent advances in investigating the potential role of a novel 3′–5′ exonuclease activity identified recently in our laboratory, a biochemical function associated, in general, with DNA repair and replication. We have employed a site-directed mutagenesis approach to demonstrate that the 3′–5′ exonuclease activity of NM23-H1 is required for its metastasis suppressor function. Consistent with a role in DNA repair, we also observe that the single yeast NM23 homolog (YNK1) is required for the maintenance of genomic integrity and normal kinetics of DNA repair in response to exposure to ultraviolet radiation. These results and their implications for understanding the molecular mechanisms underlying NM23-H1 functions in cancer are discussed.