单纯疱疹病毒I型胸苷激酶基因在大肠杆菌中的融合表达

用ＥｃｏＲ Ⅰ和Ｈｉｎｄ Ⅲ双酶切已构建的含单纯疱疹病毒Ｉ 型胸苷激酶（ ＨＳＶ１ ＴＫ） 基因的ｐ ＵＣ１８／ＴＫ 质粒，将切出的ＴＫ 基因片段克隆入原核表达载体ｐ ＷＲ４５０１ 中，构建成ＨＳＶ１ ＴＫ 基因重组表达质粒ｐ ＷＲ４５０１／ＴＫ。以ＨＳＶ１ ＴＫ 特异性引物ＴＫ１ Ｆｏｒ 和ＴＫ２ Ｒｅｖ 进行ＰＣＲ 鉴定可扩增出预期的５０３ ｂｐ 的ＴＫ 基因编码区部分序列；ＥｃｏＲ Ⅰ和Ｈｉｎｄ Ⅲ双酶切质粒ｐ ＷＲ４５０１／ ＴＫ， 可切出约１１５０ｂｐ 的ＤＮＡ 片段， 表明重组质粒中已插入ＨＳＶ１ ＴＫ基因片段。用ＩＰＴＧ 诱导ｐ ＷＲ４５０１／ＴＫ／ＪＭ１０９ ，表达出相对分子质量（ Ｍｒ） 约为９７ ０００ 的ＴＫ 和β半乳糖苷酶（ Ｍｒ５５ ０００） 融合蛋白。薄层扫描显示，该融合蛋白含量占菌体蛋白总量的２７ ．４７ ％ 。     

pLTRNL介导HSV—1 TK基因长期稳定转染人肝癌细胞系的建立

以脂质体基因转移技术，将含ＨＳＶ－１ＴＫ基因的重组逆转录病毒载体ｐＬＴＲＮＬ转染人肝癌细胞系ｈＨＣＣ，经抗生素Ｇ４１８选择，筛选出ＨＳＶ－１ＴＫ基因转染的ｈＨＣＣ再进行克隆化和亚克隆化后扩大培养，应用ＰＣＲ，ＲＴ－ＰＣＲ及ｓｌｏｔ－ｂｌｏｔ狭槽印迹等方法。对转染细胞基因组ＤＮＡ和ＲＮＡ进行了鉴定。结果表明，ＨＳＶ－１ＴＫ基因已整合入ｈＨＣＣ细胞基因组中，且有ＴＫ基因的ｍＲＮＡ转录。ＨＳＶ－１ＴＫ基     

中国流行株HIV—1gag和hIL—2／hIL—6核酸疫苗构建与实验免疫研究

目的 探讨中国流行株ＨＩＶ－１ｇａｇ与ｈＩＬ－２／ｈＩＬ－６共表达重组核酸疫苗闰的免疫效果。方法 以核酸疫苗质粒ｐＩＲＥＳ１ｎｅｏ为表达载体,构建重组核酸疫苗质料ｐＩＲＥＳ１－ｇａｇ、ｐＩＲＥＳ１－ｇａｇ－ｈＩＬ－２、ｐＩＲＥＳ１－ｇａｇ－ｈＩＬ－６,通过间接免疫荧光试验、Ｄｏｔ－ＥＬＩＳＡ检测ｇａｇ／ｈＩＬ－２／ｈＩＬ－６基因的表达产物。另将此重组核酸疫苗质粒免疫Ｂａｌｂ／ｃ小鼠,进行淋巴细胞转化试验、ＣＤ４＾＋、ＣＤ８＾＋Ｔ淋巴细胞数量测定、细胞毒性Ｔ淋巴细胞（ＣＴＬ）特异性杀伤作用检测及血清抗体检测,结果 构建的重组质粒转染ＢＨＫ细胞后可表达目的基因,免疫小鼠后可有效地刺激淋巴细胞增殖、诱导特异性ＣＴＬ反应,当和ｈＩＬ－２／ｈＩＬ－６共表达时免疫效果更加显著。讨论 与Ｇａｇ蛋白共表达的ｈＩＬ－２／ｈＩＬ－     

表达单纯疱疹病毒Ⅱ型糖蛋白D的重组痘苗病毒活疫苗株的建立

我们以前曾报道，表达单纯疱疹病毒Ⅱ型糖蛋白Ｄ（ＨＳＶ－２ｇＤ）的重组痘苗病毒（实验疫苗株）能保护被免疫小鼠抵抗致死量ＨＳＶ－２病毒的攻击。在此工作基础上，严格按人用疫苗研究要求的实验条件，成功地建立了表达ＨＳＶ－２ｇＤ的重组痘苗病毒活疫苗株。首先将经聚合酶链反应（ＰＣＲ）修饰的ＨＳＶ－２ｇＤ基因插入痘苗表达质粒ｐＪＳＢ１１７５，置于痘苗病毒Ｐ７５Ｋ早／晚期启动子控制下。将此重组质粒用Ｌｉｐｏｆｅｃｔｉｎ方法转染已受野型ＴＫ＋痘苗病毒天坛７６１株感染的人胚肺二倍体细胞。经同位素探针（３２Ｐ－ＨＳＶ－２ｇＤ）原位杂交法和３轮蚀斑纯化，筛选出基因组内整合有ＨＳＶ－２ｇＤ基因的重组痘苗病毒。斑点和Ｓｏｕｔｈｅｒｎ杂交证实，ＨＳＶ－２ｇＤ基因已插入痘苗病毒基因组内预期的ＴＫ区段，间接免疫荧光检测显示，重组病毒感染细胞后能有效地表达ＨＳＶ－２ｇＤ蛋白。     

丙型肝炎病毒及恶性疟原虫复合抗原基因表达及诱导小鼠免疫应 …

将合成的丙型肝炎病毒（ＨＣＶ）复合多表位抗原基因ＰＣＸ及恶性疟原虫（Ｐｆ）多价抗原基因ＡＢ（ＳＰｆ６６＋ＳＰｆ１０５）克隆到表达载体ｐ＾ＷＲ４５０－１中,以β－半乳糖甘酶（ＧＺ）－ＨＣＶ－Ｐｆ复合多肽抗原的形式在大肠杆菌中高效融合表达了目的蛋白ＧＺ－ＣＡＢ,表达量约３０％。所表达的重组抗原可被ＨＣＶ患者血清及鼠抗Ｐｆ抗体特异识别,显示了该融合蛋白质具有ＨＣＶ和Ｐｆ的免疫原性。该蛋白免疫小鼠后,诱发     

结核杆菌HSP70在耻垢分枝杆菌中的表达及其免疫原性研究

目的在重组分枝杆菌中表达编码人结核杆菌（ＴＢ）热休克蛋白７０（ＨＳＰ７０）的ＤｎａＫ基因，并观察其对小鼠的免疫效应。方法采用基因工程和免疫学技术将ＤｎａＫ基因及其两侧的表达调控区一起从质粒ｐＭＴ－７０中切出，经末端修饰后装入大肠杆菌－分枝杆菌穿梭质粒ｐＢＣＧ－２０００中，构建成新的重组质粒ｐＢＣＧ－ＴＢ７０，并用以转化耻垢分枝杆菌及大肠杆菌，用Ｗｅｓｔｅｒｎｂｌｏｔ检测表达的ＨＳＰ７０；以重组耻垢分枝杆菌分别经皮下及腹腔免疫小鼠，用淋巴细胞刺激指数（ＳＩ）反映细胞增殖能力，以ＮＯ法检测巨噬细胞吞噬活性，并检测血清中特异性抗ＴＢＨＳＰ７０的抗体。结果ＴＢＨＳＰ７０能在分枝杆菌中表达，表达量占菌体总蛋白量的１０％，不能在大肠杆菌中表达；重组耻垢分枝杆菌以１０６ＣＦＵ的剂量经皮下免疫小鼠后，可使小鼠脾淋巴细胞刺激指数（ＳＩ）和腹腔巨噬细胞吞噬活性增高（Ｐ＜０．０５），并能刺激机体产生特异性抗ＴＢＨＳＰ７０的抗体，腹腔免疫激发的抗体滴度较皮下免疫为低，而ＳＩ无明显改变。结论构建的表达质粒ｐＢＣＧ－ＴＢ７０能在耻垢分枝杆菌中表达ＨＳＰ７０，该重组菌具有较强的免疫原性。     

抗肾综合征出血热病毒人单克隆抗体杂交瘤细胞株的建立及初步鉴定

以亲和层析技术纯化的肾综合征出血热病毒（ＨＦＲＳＶ）结构蛋白（５５０００，６７０００）为特异性抗原，体外免疫正常人外周血淋巴细胞（ＰＢＬ），然后将体外免疫的淋巴细胞与人－鼠种间杂交瘤细胞（Ｋ６Ｈ６／Ｂ５）融合，经ＥＬＩＳＡ间接法筛选出２株（２Ｄ５，１Ｂ７）分泌抗－ＨＦＲＳＶ人单克隆抗体（Ｈ－ＭｃＡｂ）杂交瘤细胞株，４次克隆化后，１００％阳性。对２Ｄ５株初步鉴定表明，其抗体类型为ＩｇＧ１；培养上清中抗体浓度为２０～３０μｇ／ｍｌ；特异性免疫荧光反应证明２Ｄ５株Ｈ－ＭｃＡｂ是ＨＦＲＳＶ特异性；中和效价为１∶１６０；体外连续传代６个月仍稳定分泌抗体。     

检测Epstein-Barr病毒特异性细胞毒性T淋巴细胞方法的建立及其初步研究

目的建立一种非放射性、简便易行的可检测特异性细胞毒性Ｔ淋巴细胞的方法，并且初步应用于Ｅｐｓｔｅｉｎ－Ｂａｒ病毒的细胞免疫应答。方法用重组的ＥＢＶ－ＬＭＰ１痘苗病毒、ＴＫ＋痘苗病毒和杆状病毒系统表达的ＥＢＶ－ＬＭＰ１蛋白分别免疫Ｂａｌｂ／Ｃ小鼠，用Ｐ８１５细胞和乳酸脱氢酶法检测ＥＢ病毒特异性细胞毒性Ｔ细胞的杀伤效应。结果重组ＥＢＶ－ＬＭＰＩ痘苗病毒免疫组原发ＣＴＬ水平和体外诱生的二次ＣＴＬ水平均高于ＴＫ＋痘苗病毒免疫组和正常组；杆状病毒系统表达的ＥＢＶ－ＬＭＰ１蛋白免疫组的ＣＴＬ水平也明显高于正常鼠。结论本法可以较好的反映ＥＢ病毒特异性细胞毒性Ｔ细胞的水平，而且再一次说明ＬＭＰ１基因能够诱发特异性的细胞免疫。     

胸腺嘧啶核苷激酶/丙氧鸟苷自杀基因系统旁观者效应机制的探讨

目的 探讨用单纯疱疹病毒胸腺嘧啶核苷激酶／丙氧鸟苷系统进行基因治疗时发生的旁观才效应机制。方法 构建表达ＨＳＶ－ＴＫ和β－半乳糖苷酶基因的重级逆转录病毒载体,将其分别直接导入胰腺癌细胞,细胞计数检测ＧＣＶ对ＨＳＶ－ＴＫ基因转化细胞的抑制作用;以Ｌａｃ Ｚ基因转化细胞为旁观细胞,唑蓝法检测旁观者效应,并通过上清移换实验１维拉帕米抑制实验及电镜观察超微结构。结果 ＧＣＶ对ＨＳＶ－ＴＫ基因转化细胞的生长     

将抗体库所获抗-HBs Fab转换为全长人IgG

目的 将大肠杆菌表达的抗－乙肝表面抗原（ＨＢｓ）人Ｆａｂ转换为真核表达的全长人ＩｇＧ１。方法 用重叠ＰＣＲ法将人工合成的人Ｖｋ前导序列拼到抗－ＨＢｓ的ＶＨ和ＶＫ上,构建人ＩｇＧ１真核表达载体。转染真核细胞,通过ＥＬＩＳＡ、ＲＴ－ＰＣＲ、免疫印迹检测抗－ＨＢｓ人ＩｇＧ的表达。结果 用轻链和重链同时表达的单一载体在ＣＨＯ细胞中获得了抗－ＨＢｓ人ＩｇＧ的表达。结论 通过噬菌体抗体库所获Ｆａｂ段可经拼接人     

<Emphasis Type="Italic">Rana grylio</Emphasis> virus thymidine kinase gene: an early gene of iridovirus encoding for a cytoplasmic protein

The presence of thymidine kinase (TK) is a feature of many large DNA viruses. Here, a TK gene homologue was cloned and characterized from Rana grylio virus (RGV), a member of family Iridoviridae. RGV TK encodes a protein of 195 aa with a predicted molecular mass of 22.1 kDa. Homologues of the protein were present in all the currently sequenced iridoviruses, and phylogenetic analysis showed that it was much close to cellular TK type 2 (TK2), deoxycytidine kinase (dCK) and deoxyguanosine kinase (dGK). Subsequently, Western blotting revealed TK expression increased with time from 6 h post-infection in RGV-infected cells. Using drug inhibition analysis by protein synthesis inhibitor (cycloheximide) and DNA replication inhibitor (cytosine arabinofuranoside), RGV TK was classified as the early expression gene during in vitro infection. Subcellular localization by TK–GFP fusion protein expression and immunofluorescence staining showed RGV TK was an exclusively cytoplasmic protein in fish cells. Collectively, current data indicate that RGV TK was an early gene of iridovirus which encoded a cytoplasmic protein in fish cells. The sequence reported here has been deposited in the GenBank database under accession no EU747722.     

Human papillomavirus type 16 E7 protein expressed in Escherichia coli and monkey COS-1 cells: immunofluorescence detection of the nuclear E7 protein

Human papillomavirus type 16 E7 protein, expressed in Escherichia coli as fusion protein lac alpha peptide-E7 (lac-E7), was purified by electrophoresis and used to immunize rabbits to raise antisera. The anti-lac-E7 sera recognized another bacterially expressed fusion protein trpE-E7 by the Western blot method. The antisera were used to identify E7 protein transiently expressed in monkey COS-1 cells from an SV40-derived expression plasmid containing E7 gene. Like the E7 protein from CaSki cells, the majority of 19K E7 protein in the transfected COS-1 cells was found by immunoprecipitation in the soluble cytoplasmic fraction. By immunofluorescence staining, however, the E7 protein was detectable in the nuclei of the transfected COS-1 cells. The results suggest that the E7 protein expressed in monkey cells is nuclear, although it is readily released from nuclei when the cell structure is broken.     

Notch信号途径效应分子HES1的原核表达及其多克隆抗体的制备与鉴定

目的 原核表达HES1分子，并制备其特异性多克隆抗体，以研究该分子在肿瘤细胞中的表达情况。方法 诱导表达GST-HES1融合蛋白和GST，经谷胱甘肽-Sepharose4B亲和纯化后，用GST偶联预激活的Sepharose4B，以GST-HES1为免疫原制备兔抗血清，得到的抗血清经GST-Sepha-rose4B吸收抗GST成分，以间接ELISA和Western blot鉴定抗本特异性，以Western blot分析胃癌，结肠癌及其相应非癌变组织中HES1的表达水平。结果 成功地表达并纯化了GST-HES1融合蛋白，制备的抗HES1多克隆抗体具有很高的特异性，与GST或大肠杆菌成分无交叉反应，用于进行天然HES1分子的Wistern blot检测时效果良好，初步检测发现，胃癌和结肠癌中HES1的表达水平明显高于相应的正常组织。结论 通过原核表达并纯化HES1，成功地制备了该分子的特异性多克隆抗体，初步应用效果满意。     

兔抗人PON2抗体的制备与初步应用

目的:制备兔抗人PON2(paraoxonase-2)的多克隆抗体并进行初步鉴定.方法:生物信息学方法分析人PON2蛋白序列,选取人兔同源性较低,亲水性及免疫原性较强的片段,通过大肠杆菌原核表达系统进行重组表达,获得GST-PON2融合蛋白用于免疫新西兰大耳白兔获得多克隆抗体,通过XX方法分离纯化得到的抗PON2多克隆抗体,用West-ern blot、间接免疫荧光对其进行特异性及灵敏度鉴定.结果:成功获得了高表达的相对分子质量(Mr)为46 000的PON2重组融合蛋白;Western blot鉴定结果显示此多克隆抗体可特异识别肝脏总蛋白、HeLa细胞及U937细胞中Mr为39 000的天然PON2蛋白;间接免疫荧光结果显示此多克隆抗体所识别的蛋白定位于SY5Y细胞胞质中.结论:抗人PON2的多克隆抗体特异识别肝总蛋白、HeLa细胞及U937细胞中的天然蛋白,可用于PON2的研究及临床检测.     

人睾丸精子结合蛋白-His6融合蛋白在真核细胞中的表达及亲和纯化

目的:构建含有人睾丸精子结合蛋白基因(testisspermbindingprotein,tsbp)的真核表达载体pcDNA3.1/mycHis(-)B/tsbp,并进行表达和纯化。方法:以克隆有tsbp全长cDNA序列的原核表达载体pGEX5X1/tsbp为模板,用PCR扩增tsbp,并通过DNA重组构建真核表达载体pcDNA3.1/mycHis(-)B/tsbp。以重组体稳定转染HEK293细胞后,应用RTPCR、Westernblot和细胞免疫荧光技术检测His6tsbp融合蛋白的表达。选择高表达量的细胞克隆,扩增并用Ni2 NTA金属亲和层析柱(IMAC)从细胞裂解液中纯化融合蛋白His6tsbp,纯化产物的纯度用SDSPAGE和Westernblot进行鉴定。结果:成功地构建了真核表达载体pcDNA3.1/mycHis(-)B/tsbp。以重组体转染HEK293细胞后,用RTPCR检测到tsbpmRNA的表达。细胞免疫荧光染色呈阳性反应,Westernblot检测到培养细胞中有融合蛋白His6tsbp的表达。经Ni2 NTA金属亲和层析柱分离纯化,得到纯化的重组蛋白His6tsbp。结论:构建了pcDNA3.1/mycHis(-)B/tsbp真核表达载体,并在HEK293细胞中表达和亲和层析纯化,为下一步的研究奠定了基础。     

Production and characterisation of a novel chicken IgY antibody raised against C-terminal peptide from human thymidine kinase 1

Egg yolk is a good source of highly specific antibodies against mammalian antigens because of the phylogenetic distance between birds and mammals. Chicken egg yolk immunoglobulins (IgY) were generated to a synthetic 31-amino acid peptide from the C-terminal of human HeLa thymidine kinase 1 (TK1) enzyme. The anti-TK1 IgY antibody was purified using affinity chromatography against the 31-amino acid peptide. The purified antibody inhibited the catalytic activity of the TK1 enzyme in the CEM TK1(+) cells and recognized the 25-kDa subunit and tetrameric form of TK1, which has a pI value of 8.3. No immunoreaction was observed in CEM TK1(-) cells. Western blot of the serum TK1 (S-TK1) also showed that only a single band was found in the serum of patients with malignancies. No band was seen in healthy serum. Furthermore, dot blots and enhanced chemiluminescence (ECL) detection of S-TK1 performed on sera of preoperative patients with gastric cancer (GC) (n=31) and healthy controls (n=62) showed that the levels of S-TK1 in the sera of cancer patients were significantly different (P<0.01). Using ECL dot blots, 0.1 pg of TK1 in 3 microl sera could be detected. Immunohistostaining of tissues in the 11 advanced-stage cancer patients (four breast carcinomas, three hepatocarcinomas and four thyroid carcinomas) indicated that a strong staining of TK1 enzyme was found in the cytoplasm of malignant cells. No staining or weak staining was seen in normal tissues. We suggest that screening for TK1 using anti-TK1 IgY may be potentially useful for serological and immunohistochemical detection of TK1 as an early prognosis and for monitoring patients undergoing treatment.     

F10蛋白的原核表达及多克隆抗体的制备

目的：表达F10蛋白，并制备兔抗F10多克隆抗体。方法：利用PCR方法扩增F10基因片段，经BamHⅠ和EcoRⅠ酶切后连接人pET-GST原核表达载体，构建的pET-GST／F10融合重组表达质粒转化大肠杆菌B121，经IPTG诱导表达蛋白，并以亲和层析的方法进行纯化。表达产物用SDS—PAGE电泳和Western blot进行分析鉴定。以纯化的F10蛋白免疫新西兰大白兔，制备兔抗F10的多克隆抗体，并以ELISA法检测抗体效价。结果：经酶切和核酸序列分析证实重组质粒包含有正确编码的F10读码框。SDS—PAGE电泳分析显示pET—GST／F10诱导后表达一相对分子质量（Mr）约为61000的融合蛋白，与预期结果相符。目的蛋白纯化后的纯度达90％以上，Western blot证实该蛋白是GST／F10的融合蛋白。将纯化的GST／F10融合蛋白免疫家兔，得到的兔抗F10抗体效价达1：20000。结论：成功构建了人F10基因原核表达载体，并获得了高纯度的F10重组蛋白及兔抗F10抗体，为下一步研究F10基因功能奠定了实验基础。     

Development of a Western blot assay for detection of bovine immunodeficiency-like virus using capsid and transmembrane envelope proteins expressed from recombinant baculovirus

A 120-amino-acid polypeptide selected from the transmembrane protein region (tTM) and the major capsid protein p26 of bovine immunodeficiency-like virus (BIV) were expressed as fusion proteins from recombinant baculoviruses. The antigenic reactivity of both recombinant fusion proteins was confirmed by Western blot with bovine and rabbit antisera to BIV. BIV-negative bovine sera and animal sera positive for bovine syncytial virus and bovine leukemia virus failed to recognize the recombinant fusion proteins, thereby showing the specificity of the BIV Western blot. One hundred and five bovine serum samples were tested for the presence of anti-BIV antibodies by the recombinant protein-based Western blot and a reference Western blot assay using cell culture-derived virions as test antigens. There was a 100% concordance when the p26 fusion protein was used in the Western blot. However, the Western blot using the tTM fusion protein as its test antigen identified four BIV-positive bovine sera which had tested negative in both the p26 recombinant-protein-based and the reference Western blot assays. This resulted in the lower concordance of 96.2% between the tTM-protein-based and reference Western blot assays. The results of this study showed that the recombinant p26 and tTM proteins can be used as test antigens for the serodetection of BIV-infection in animals.     

Development of a Western Blot Assay for Detection of Bovine Immunodeficiency-Like Virus Using Capsid and Transmembrane Envelope Proteins Expressed from Recombinant Baculovirus

A 120-amino-acid polypeptide selected from the transmembrane protein region (tTM) and the major capsid protein p26 of bovine immunodeficiency-like virus (BIV) were expressed as fusion proteins from recombinant baculoviruses. The antigenic reactivity of both recombinant fusion proteins was confirmed by Western blot with bovine and rabbit antisera to BIV. BIV-negative bovine sera and animal sera positive for bovine syncytial virus and bovine leukemia virus failed to recognize the recombinant fusion proteins, thereby showing the specificity of the BIV Western blot. One hundred and five bovine serum samples were tested for the presence of anti-BIV antibodies by the recombinant protein-based Western blot and a reference Western blot assay using cell culture-derived virions as test antigens. There was a 100% concordance when the p26 fusion protein was used in the Western blot. However, the Western blot using the tTM fusion protein as its test antigen identified four BIV-positive bovine sera which had tested negative in both the p26 recombinant-protein-based and the reference Western blot assays. This resulted in the lower concordance of 96.2% between the tTM-protein-based and reference Western blot assays. The results of this study showed that the recombinant p26 and tTM proteins can be used as test antigens for the serodetection of BIV-infection in animals.