重组人干扰素Epsilon的表达纯化及生物学性质研究

目的表达纯化一种新型重组人干扰素Epsilon(rhIFNε155ser),并对它的生物学特性进行初步研究。方法人工合成rhIFNε155ser的全基因序列,并按照大肠埃希菌密码子嗜性作适当改造,然后构建到原核表达载体pBV220,在大肠埃希菌DH5α中表达。将表达的包涵体蛋白纯化复性后,对最终产物的抗病毒活性、细胞生长抑制活性和NK细胞刺激活性进行鉴定。同时,利用芯片技术,从基因的水平对rhIFNε155ser的生物机制进行初步的了解。结果rhIFNε155ser以包涵体的形式表达,经纯化蛋白纯度可达95%。复性后rhIFNε155ser在WISH抗VSV系统中的抗病毒活性可达6×105IU/mg。在100～1000pg/ml下rhIFNε155ser具有剂量依赖性的抗增殖活性,而NK细胞刺激活性无剂量依赖性。基因芯片扫描发现,22278个位点中,有283个基因显著上调,另外1894个显著下降。结论成功地表达了rhIFNε155ser,发现此IFN具有一定的抗病毒、抗增殖和NK细胞刺激活性。芯片技术可进一步拓展对IFN的功能研究。     

IFN-λ在人食管癌细胞系中诱导的生物学活性的初步研究

目的 初步研究IFN-λ在7种人食管癌细胞系中诱导的生物学作用。方法 PCR检测IL-28α和IL-10β的基因及抗病毒分子基因的表达,流式细胞术检测主要组织相容性抗原的表达及细胞周期,二苯基溴化四氮唑蓝(MTT)检测细胞增殖。结果 各食管癌细胞表达IL-28α和IL-10β的基因;IFN-λ诱导或上调2′5′-寡腺苷酸合成酶（2′5′-OAS）和黏病毒抗性蛋白A(MxA)的基因表达;IFN-λ可上调Ⅰ类主要组织相容性抗原分子的表达;IFN-λ可通过调控细胞周期的方式抑制食管癌细胞生长增殖。结论 人食管癌细胞株表达IFN-λ受体复合体,IFN-λ具有抗病毒、抗增殖和免疫调节的生物学活性。     

人IFN-ε的表达、纯化及生物学特性研究

目的 表达并纯化人IFN-ε,并对其理化特性和生物学特性进行初步研究. 方法 利用PCR技术以人基因组DNA为模板,获得目的片段的全基因序列后,克隆到原核表达载体pET-32a(+)上,构建重组表达载体pET-32a(+)/IFN-ε,转化到大肠杆菌BL21(DE3)中进行表达,并对表达进行了优化.产物的表达形式为包涵体,经过纯化复性后,获得高纯度的活性蛋白IFN-ε.对IFN-ε的理化性质和抗病毒活性、抑制肿瘤细胞增长活性、刺激细胞产生抗病毒蛋白的活性进行了鉴定. 结果 IFN-ε以包涵体形式表达,纯化后纯度大于95%,抗病毒活性为1.2×105 IU/mg,能够抑制肿瘤细胞的生长,并能诱导细胞产生抗病毒蛋白MxA. 结论 成功表达了人IFN-ε蛋白,并证明此蛋白质具有抗病毒和抗细胞增殖的活性.     

干扰素λ1在CHO细胞中的表达

目的 构建干扰素λ1表达载体PCI-dhfr-λ1和连接增强子SP163的表达载体PCI-dhfr-SP163-λ1,并在CHO(dhfr-)细胞中表达.方法 在合成的干扰素λ1基因中引入相应酶切位点,并通过融合PCR获得连接增强子SP163-λ1基因,测序正确后将该基因插入表达载体PCI-dhfr,构建重组表达载体PCI-dhfr-λ1和PCI-dhfr-SP163-λ1.将所构建的重组表达载体用脂质体法转入CHO(dhfr-)细胞,通过间接免疫荧光法和Western Blot鉴定了λ1蛋白的表达,应用细胞病变抑制法初步鉴定了λ1蛋白的抗病毒活性.结果 成功构建了干扰素λ1真核表达载体PCI-dhff-λ1和PCI-dhfr-SP163-λ1.免疫荧光结果显示分别转染了两种表达载体的CHO(dhfr-)细胞均能表达干扰素λ1蛋白,SP163增强子明显增强了干扰素λ1蛋白的表达,Western Blot结果显示转染SP163的CHO(dhfr-)细胞表达干扰素λ1蛋白.通过细胞病变抑制实验,在转染细胞48h后的细胞培养液中检测到了干扰素λ1的抗病毒活性.结论 本研究成功的在CHO(dhfr-)细胞中表达了干扰素λ1,表达的蛋白具有抗病毒活性,为进一步建立稳定表达干扰素λ1的细胞系提供了条件.     

人Epsilon干扰素在CHO细胞中的表达及生物学活性的研究

目的 为了研究新发现的人epsilon干扰素（hIFN-ε）的生物学活性，我们通过RT-PCR克隆了hIFN-ε基因，并构建pcDNA3．1／myc-his（-）A-hIFN-ε（以下简称pcDNA3-1A-hIFN-ε）的真核表达载体，在CHO细胞中表达，并研究真核细胞重组表达的rhIFN-ε的生物学活性。方法 用TNF-α刺激人宫颈癌HeLa细胞，抽提总RNA，经RT-PCR获得全长cDNA，与pcDNA3．1／myc-his（-）A真核表达载体连接，构建重组体pcDNA3．1A-hIFN-ε并在CHO细胞中进行表达，采用微量细胞病变抑制试验研究表达产物中的rhIFN-ε对多种病毒的抗病毒活性；MTT法检测其对A375、HeLa和A549细胞的生长影响，并通过在Wish、HeLa、A375细胞中诱导MxA抗病毒蛋白的产生研究hIFN-ε的抗病毒机制。结果 经PCR和限制性酶切鉴定以及DNA测序，结果表明已经成功构建了重组体pcDNA3．1A-hIFN-ε，并在CHO细胞中稳定表达了rhIFN-ε蛋白，该蛋白具有抗HSV-I、PolioV、Ad3和VSV病毒的作用，能够抑制HeLa、A375、A549细胞的生长，刺激Wish、HeLa、A375细胞产生MxA蛋白。结论 本研究成功地构建了pcDNA3．1A-hIFN-ε真核表达载体并在CHO细胞中获得稳定表达，证明了ddFN．￡蛋白具有抗病毒和抗增殖活性，其抗病毒效应的分子机理可能与诱导细胞产生MxA抗病毒蛋白有关，为今后研究rhIFN-ε的生物学功能和基因重组药物研制及其开展基因治疗奠定了基础。     

土拨鼠α干扰素新亚型基因的克隆及生物学活性分析

目的 克隆土拨鼠α干扰素（IFN-α）新亚型基因，用于土拨鼠HBV模型探索IFN-α治疗慢性乙型肝炎策略；调查慢性土拨鼠肝炎病毒（woodchuck hepatitis virus，WHY）感染的土拨鼠外周血单个核细胞（PBMC）干扰素功能状况。方法 poly（I-C）体外刺激正常和慢性WHV感染的土拨鼠PBMC，分析其表达的干扰素生物学活性。利用分子克隆技术对土拨鼠IFN-α家族基因进行克隆，并对所克隆的系列基因进行测序、分型并进行真核表达后检测表达产物生物学活性。结果 poly（I-C）刺激体外培养的土拨鼠PBMC后，慢性感染土拨鼠PBMC分泌的干扰素的活性显著差异低于正常土拨鼠（P〈0．01）。获得36个土拨鼠IFN-α基因序列克隆，测序分析后，发现有10个克隆是新亚型基因，其中8个为功能基因亚型，2个为假基因亚型，病毒保护试验证明只有功能基因亚型具有生物学活性。结论 慢性WHV感染的土拨鼠细胞免疫功能受损。新的土拨鼠IFN-α亚型基因的克隆为在土拨鼠HBV动物模型上进行干扰素基因治疗和研究干扰素治疗策略提供了新的材料。     

重组人ω-干扰素的制备及活性分析

目的：建立人ω-干扰素（hIFN-ω）的大肠杆菌表达系统，为人ω-干扰素的生产及临床应用奠定基础。方法：自抗凝血中提取人基因组DNA，PCR扩增人ω-干扰素成熟肽编码序列克隆入pGEM—T—easy载体，进一步构建表达型重组质粒pBV-IFN-ω，温度诱导表达，表达产物行SDS-PAGE及Western blot鉴定。溶解并复性包涵体，细胞病变抑制法测定表达产物的抗病毒活性。结果：DNA序列分析证实，重组质粒pBV-IFN-ω含有开放读码框架正确的人ω-干扰素成熟肽编码序列。SDSPAGE显示，诱导表达碎菌后的沉淀中有大小约为20kD的外源蛋白，Western blot证明此外源蛋白为重组人ω-干扰素，并经体外活性测定证实有良好的抗病毒活性。结论：成功地构建了人ω-干扰素（IFN-ω）的大肠杆菌表达系统，表达出具有抗病毒活性的重组人ω-干扰素。     

鸡MxA基因的克隆、表达及生物学活性检测

目的：克隆鸡MxA基因，构建其原核表达质粒，并诱导其在大肠杆菌中表达融合蛋白。方法：采用RT-PCR方法从鸡成纤维细胞（CEF）中扩增MxA，将其克隆至克隆载体pMD18-T中，筛选阳性克隆后回收目的片段，将其克隆人原核表达质粒pGEX-6p—1，构建其重组表达质粒pGEX—MxA，以IPTG诱导表达，经SDS—PAGE、鸡胚新城疫病毒干扰试验和VSV-CEF微量细胞抑制试验进行分析、鉴定。结果：经RT—PCR扩增获得的MxA序列与GeneBank报道的序列一致，SDS—PAGE和干扰试验证实重组质粒可以表达出相应分子量为45000的蛋白，与GST—MxA融合蛋白分子量一致。结论：成功完成了鸡MxA基因的克隆及其原核表达质粒的构建，在大肠杆菌DIL5ct中经IPTG诱导表达了融合蛋白GST—MxA，为进一步探讨MxA的生物学活性，探索抗病毒药物的研发奠定了基础。     

鼠IFN-λ2 CHO细胞系建立及生物学活性的研究

目的 稳定表达鼠IFN-λ2并对其生物学活性进行研究.方法 用水疱口炎病毒(vesicular stomatitis virus,VSV)刺激小鼠脾脏细胞,克隆mIFN-λ2全长基因,构建真核表达载体PCAGG-EGFP-mIFN-λ2,并在CHO细胞稳定表达,且在小鼠黑色素瘤B16细胞上进行抗病毒活性测定;构建MDBK-Mxp-Luc细胞系诱导Mx1抗病毒蛋白产生.结果 pMD18-T-mIFN-λ2双酶切鉴定,出现582 bp大小的条带,成功构建了PCAGG-EGFP-mIFN-λ2真核表达载体;稳定表达mIFN-λ2 CHO的细胞株分泌的上清中mIFN-λ2蛋白在B16细胞上的抗病毒活性为10~4 AU/ml;mIFN-λ2蛋白诱导鼠Mx1抗病毒蛋白的表达,9～12 h达高峰,24 h后消失(P<0.05).结论 建立了稳定表达mIFN-λ2的CHO细胞株,其分泌型mIFN-λ2蛋白具有明显的抗病毒活性,且与诱导Mx1抗病毒蛋白密切相关.     

复合干扰素在毕赤酵母中的分泌表达、纯化及活性分析

目的 在毕赤酵母中高效分泌表达复合干扰素。方法 根据毕赤酵母密码子偏性合成了复合干扰素基因，克隆至分泌型酵母表达载体pMKX9K，线性化后转化毕赤酵母GS115，筛选高表达菌株。诱导后的培养上清经过离子交换，疏水层析，凝胶过滤三步层析纯化，用细胞病变抑制法测定其活性，并结合Iowry法蛋白定量计算其比活性。结果 SDS-PAGE分析显示，重组蛋白以可溶性分子的形式存在于上清液中，经纯化后纯度达到96％，其N端氨基酸序列与理论值一致，比活与干复津相当。结论 复合干扰素在毕赤酵母中获得高效分泌表达，为获得大量基因工程产品奠定了基础。     

Persistence of Sindbis virus in BHK-21 cell cultures

Summary If BHK-21 cell monolayer cultures are inoculated with Sindbis virus individual surviving cells can be observed, which are capable to multiply thereby forming small colonies in which the virus persists. The virus causes periodical cell destructions usually starting in the centers of the colonies. Increasing virus titers are observed during the phases of cell destruction and decreasing titers during the phases of cell growth. It takes several weeks for the slow growing colonies to form confluent cell sheets. Those cultures can be trypsinized and grown in continuous passages. The above mentioned periodical cell destructions then do no longer occur because a more stable equilibrium between BHK-21 cells and Sindbis virus has been established. About 6% of the cells are virus producers as evidenced by infective center tests. The supernatant contains approximately 10⁴ to 10⁵ PFU per 0,1 ml. The behaviour of the cells is changed concerning plaque formation by Sindbis virus if the persistent infection is eliminated by addition of immune serum. Cell morphology, however, remains unchanged. Virus from persistently infected cells differs also from the original virus: it produces smaller plaques in BHK-21 cell and chick embryo fibroblast cultures.Partly presented at 3. Arbeitstagung der Deutschen Gesellschaft für Hygiene und Mikrobiologie, Mainz, October 8–10, 1970.     

Expression of sarcomeric proteins and assemblyof myofibrils in the putative myofibroblast cell line BHK-21/C13

The expression and organization patterns of several myofibrillar proteins were analysed in the putative myofibroblast cell line BHK-21/C13. Although this cell line originates from renal tissue, the majority of the cells express titin. In these cells, titin is, under standard culture conditions, detected in myofibril-like structures (MLSs), where it alternates with non-muscle myosin (NMM). Expression of sarcomeric myosin heavy chain (sMyHC) is observed in a small minority of cells, while other sarcomeric proteins, such as nebulin, myosin binding protein C (MyBP-C), myomesin and M-protein are not expressed at all. By changing the culture conditions in a way equal to conditions that induce differentiation of skeletal muscle cells, a process reminiscent of sarcomerogenesis in vitro is induced. Within one day after the switch to a low-nutrition medium, myofibrillar proteins can be detected in a subset of cells, and after two to five days, all myofibrillar proteins examined are organized in typical sarcomeric patterns. Frequently, cross-striations are visible with phase contrast optics. Transfection of these cells with truncated myomesin fragments showed that a specific part of the myomesin molecule, known to contain a titin-binding site, binds to MLSs, whereas other parts do not. These results demonstrate that this cell line could serve as a powerful model to study the assembly of myofibrils. At the same time, its transfectability offers an invaluable tool for in vivo studies concerning binding properties of sarcomeric proteins. © Kluwer Academic Publishers.     

Influenza and mumps diagnostic antigens from BHK-21 cell cultures

Summary Crude CF antigens for serological diagnosis of infections with influenza type A, type B or mumps were readily prepared from the cellular phase of infected BHK-21 cell cultures. These antigens were mainly soluble. When treated with 2% chloroform they gave similar titres with human convalescent sera as the corresponding egg-derived antigens.     

人白细胞介素21在真核细胞中表达及其体外促T细胞增殖作用

目的：构建人细胞因子IL-21表达载体并在Hela细胞中稳定表达，分析rhIL-21体外促T细胞增殖作用。方法：以已构建的pMD-1121为模板，PCR扩增成熟IL-21 cDNA的编码框序列并构建到真核表达载体pCDNA3．1／hismye-B中。挑出阳性克隆进行扩增，脂质体转染入Hela细胞中并以G418加压筛选。有限稀释法、RT-PCR及Western blot筛选出稳定表达的细胞株。培养上清液经金属离子螯合层析分离纯化后，SDS-PAGE、Westem blot鉴定。MIT法测定其与抗CD3单克隆抗体共刺激T细胞增殖活性。结果：SDS-PAGE、Western blot显示Mr为18000的带有myc重组人IL-21融合蛋白(rhIL-21)；并成功地获得hIL-21稳定表达细胞株。rhIL-21融合蛋白具有与抗CD3抗体共刺激T细胞增殖作用。结论：获得具有生物学活性的rhIL-21细胞因子，为进一步研究其功能奠定基础。     

辛德毕斯病毒结构蛋白基因对宿主细胞影响的初步研究

目的 了解辛德毕斯病毒基因结构与功能的关系,阐明其分子致病分子机理。方法 构建了辛德毕斯病毒（ＸＪ－１６０病毒株）结构基因的真核表达质粒,ｐｃＤＮＡ３．１ＡＢＣ,转染ＢＨＫ－２１细胞后观察其对宿主细胞的影响。结果 转染病毒结构基因的ＢＨＫ－２１细胞,可以发生辛协毕斯病毒感染所出现的细胞毒性反应,表现为：细胞存活率降低;细胞核染色质浓缩、凝集;Ｇ１期细胞减少、而处于Ｓ期的细胞增多。结论 辛德毕斯病毒     

Formation of wesselsbron virus in BHK-21 cells

Summary The multiplication of attenuated and virulent strains of Wesselsbron virus in BHK-21 cells have been investigated electron microscopically. In thin sections of cells infected with virulent virus and harvested at different time intervals, mesh-like structures, similar to those reported by previous workers, are observed. However, cells infected with the attenuated form of the virus do not show these structures at all, indicating that these mesh-like bodies are not a prerequisite for virus production. Cytoplasmic inclusions containing dense nucleoids and developing virus particles are reported. Mature virus particles are commonly found in cisternae of the endoplasmic reticulum in both virulent and attenuated forms of the virus. Particle measurements have yielded a diameter of 45 m, which is somewhat larger than measurements obtained by previous workers.The similarity between the mesh-like structures reported in this work, the granular virus-forming areas reported bySoutham et al. (1964) in HBp2 cells infected with West Nile virus and the structures occurring in cells infected with yellow fever virus (Bekgold andWeibbl, 1962) may indicate a close cytopathic relationship between these group B arboviruses.     

Safety evaluation of <Emphasis Type="Italic">Acalypha wilkesiana</Emphasis> in albino rats and BHK-21 cell line

Acalypha wilkesiana is a member of the spurge family, genus Acalypha, that is widely used in folklore medicine. The aim of this study was to screen aqueous leaf extract of A. wilkesiana for toxic effects in vitro and in vivo. We examined the phytochemical profile, cytotoxic effects on baby hamster kidney cell line (BHK-21), and oral subacute toxicity of A. wilkesiana leaf decoction in rats. Rats were given 0, 300, 600, and 1200 mg/kg body weight of A. wilkesiana leaf extract, daily, orally for 14 days. The phytochemical profile showed the presence of flavonoids, saponin, cardiac glycosides, and tannins. It caused apoptosis in BHK-21 cell line at concentrations of 0, 25, 50, 100, and 200 μg/ml. There was significant increase in the levels of serum AST, ALT, creatinine, urea, Na⁺, K⁺, and Cl^? levels in all the test groups compared to the control. Histology of the liver revealed centrilobular degeneration and necrosis with sinusoidal dilatation as well as polymorphonuclear and mononuclear infiltration. The kidney showed severe glomerular and tubular degeneration and necrosis with hemorrhage at all doses administered. We conclude that the plant was toxic at the doses tested in vitro and in vivo, and care should be exercised in its use in herbal medicine.