荧光定量PCR检测TCR VγI-Jγ基因重排标准品质粒及标准曲线的构建

目的：构建荧光定量PCR检测TCR VγI-Jγ基因标准品重组质粒和标准曲线，为建立实时荧光定量PCR准确检测淋巴系肿瘤微小残留病奠定基础。方法：对TCR VγI-Jγ基因进行序列分析，利用引物设计软件设计出一对引物和一条荧光探针。提取急性淋巴细胞白血病患者的DNA，经PCR扩增，产物纯化后与pMD 18-T Vector连接，转化到大肠杆菌DH5α，筛选得到标准品的质粒。结果：重组质粒进行荧光定量PCR扩增出现强烈的荧光值增长，表明TCR VγI-Jγ基因已成功克隆。以10^0～10^-5不同稀释水平的标准品进行荧光定量PCR扩增，Ct值分别为21．08、24．34、27．53、30．58和33．25。统计学分析显示Ct值与标准品浓度的对数存在良好线性关系，回归系数为0．998。结论：所构建的TCR VγI-Jγ基因荧光定量PCR检测标准品特异性和线性关系好，准确可靠，利于统一标准。     

携带绿色荧光蛋白基因的逆转录病毒载体的构建及其介导的T细胞基因转移研究

为了构建含绿色荧光蛋白（green fluorescent protein，GFP）基因的逆转录病毒载体和研究逆转录病毒对T细胞的感染能力，利用亚克隆技术将磷酸甘油酸激酶启动子（phosphoglycerate kinase promoter，PGK）基因和GFP全长cDNA插入逆转录病毒载体pLXSN，采用磷酸钙沉淀法将重组载体转染PA317包装细胞，G418筛选出抗性克隆，收集滴度最高的病毒上清感染NIH3T3和T细胞，在倒置荧光显微镜下观察GFP表达情况。结果表明；重组逆转录病毒载体转染PA317包装细胞后，可在荧光显微镜下观察到GFP的表达。G418筛选后，含GFP的逆转录病毒可感染原代培养的T细胞。结论：逆转录病毒载体能够快速、稳定地将外源基因转移至T细胞，可作为介导T细胞基因转移的重要工具。     

结核菌抗原基因Rv3881c在大肠杆菌中的高效表达分析

目的：从结核分枝杆菌基因组克隆Rv3881c基因并重组入pET24b载体，在大肠杆菌中进行融合表达。重组蛋白经纯化后通过Western blot分析初步鉴定其抗原性和特异性。方法：提取H37Rv标准株的基因组DNA。以基因组DNA为模板PCR扩增出Rv3881c基因，连接到pGEMT上。经过筛选鉴定后。将重组子测序；将测序正确的基因双酶切后连接到pET24b载体上，筛选得到重组载体pET24b-48；在BL21菌株中优化表达并采用金属鏊合层析方法纯化蛋白：选取6例结核病阳性临床血清标本和6例健康人血清标本进行Western blot分析。结果：发现从保存的H37Rv菌株克隆得到了读码框完全正确的Rv3881c基因。它在大肠杆菌中可以高效表达。表达量约占细菌总蛋白的20％以上。重组蛋白不与健康人血清反应。可与结核标本血清有较强的免疫印迹反应。结论：重组Rv3881c具有较好的特异性。该抗原有潜力成为检测结核的有效抗原组分之一。     

建立永生化皮肤成纤维细胞的初步研究：pIRES2-EGFP-hTERT真核表达载体的构建

目的：构建同时含有荧光蛋白表达基因和端粒酶催化亚单位(hTERT)表达基因的真核质粒载体，为hTERT cDNA的稳定转染提供快速有效的鉴定手段，并为建立永生化的皮肤成纤维细胞，为皮肤试验提供稳定的细胞系奠定基础。方法：选取带有荧光蛋白表达基因的空载体和带有hTERT cDNA的载体，利用基因重组技术，获得新的载体pIRES2-EGFP-hTERT，筛选阳性克隆进行酶切和序列测定。结果：酶切电泳分析，获得了3．4kb和5．3kb大小的两条片段；测序结果两端序列正确。结论：重组载体pIRES2-EGFP-hTERT构建成功,为hTERT cDNA的稳定转染提供快速有效的鉴定手段，为建立永生化的皮肤成纤维细胞系奠定了基础。     

延胡索酰乙酰乙酸水解酶基因敲除大鼠胚胎干细胞株的建立

背景：小鼠肝损伤模型已被广泛应用,大鼠肝损伤模型能在发病机制,肝移植环境等方面更好地模拟人,建立大鼠延胡索酰乙酰乙酸水解酶（fuarylacetoacetat hydrolase,Fah）基因敲除的胚胎干细胞株是大鼠肝损伤模型建立的重点和难点。目的：建立Fah基因缺失的大鼠胚胎干细胞株。方法：根据Genbank检索出的大鼠Fah基因序列构建带有正负双向筛选系统的Fah基因敲除载体pKO-Fah,并进行大鼠胚胎干细胞制备与培养,通过电穿孔转染将线性化质粒转入大鼠胚胎干细胞中,用嘌呤霉素和增强型绿色荧光进行克隆的筛选,再对筛选出来的阳性克隆进行PCR验证。结果与结论：电穿孔转染后观察到绿荧光胚胎干细胞克隆,经过3轮嘌呤霉素药物筛选后约有90%的胚胎干细胞克隆表达绿荧光,成功挑取到2个稳定表达绿荧光的胚胎干细胞克隆,重组胚胎干细胞-Fah,通过PCR鉴定为正确同源重组的阳性克隆。提示大鼠胚胎干细胞稳定建系后,能通过传统的同源重组方法来建立基因敲除胚胎干细胞,并为后期基因敲除动物的建立奠定基础。     

延胡索酰乙酰乙酸水解酶基因敲除大鼠胚胎干细胞株的建立

背景:小鼠肝损伤模型已被广泛应用,大鼠肝损伤模型能在发病机制,肝移植环境等方面更好地模拟人,建立大鼠延胡索酰乙酰乙酸水解酶(fuarylacetoacetat hydrolase,Fah)基因敲除的胚胎干细胞株是大鼠肝损伤模型建立的重点和难点。目的:建立Fah基因缺失的大鼠胚胎干细胞株。方法:根据Genbank检索出的大鼠Fah基因序列构建带有正负双向筛选系统的Fah基因敲除载体pKO-Fah,并进行大鼠胚胎干细胞制备与培养,通过电穿孔转染将线性化质粒转入大鼠胚胎干细胞中,用嘌呤霉素和增强型绿色荧光进行克隆的筛选,再对筛选出来的阳性克隆进行PCR验证。结果与结论:电穿孔转染后观察到绿荧光胚胎干细胞克隆,经过3轮嘌呤霉素药物筛选后约有90%的胚胎干细胞克隆表达绿荧光,成功挑取到2个稳定表达绿荧光的胚胎干细胞克隆,重组胚胎干细胞-Fah,通过PCR鉴定为正确同源重组的阳性克隆。提示大鼠胚胎干细胞稳定建系后,能通过传统的同源重组方法来建立基因敲除胚胎干细胞,并为后期基因敲除动物的建立奠定基础。     

重组人凝血因子Ⅶ在CHO细胞中的稳定表达、纯化及功能鉴定

目的：真核表达及纯化重组人凝血因子Ⅶ（rFⅦ）。方法通过PCR方法扩增FⅦ全长cDNA,克隆入真核表达载体pcDNA3.0,利用脂质体将FⅦ基因与二氢叶酸还原酶基因共转染CHO-dhfr-/-细胞,采用氨甲喋呤药物加压,Western blot方法筛选获得稳定高表达细胞克隆。将高表达细胞克隆在摇瓶中无血清悬浮驯化培养,收集细胞培养上清,通过离子交换和亲和层析两步法进行纯化并对纯化产物进行活性鉴定。结果在CHO细胞中成功表达了rFⅦ,无血清悬浮培养条件下表达量可达3 mg·L-1·d-1,成功纯化了rFⅦ,SDS-PAGE凝胶电泳显示分子质量约50 kDa的单一多肽,生物学活性试验证实该纯化产物具有促凝活性。结论成功表达并纯化了rFⅦ,为进一步研制活化凝血因子Ⅶ药物奠定了基础。     

结核分支杆菌16000抗原基因在大肠杆菌中的高效表达及其产物

目的：获得高效表达结核分支杆菌16000抗原的大肠杆菌工程菌，将培养物纯化后得到重组16000蛋白抗原纯品，并检测其免疫学特性。方法：用聚合酶链反应（PCR）方法获得目的基因构建大肠杆菌高效表达株。聚丙烯酰胺凝胶电泳（SDS—PAGE）分析重组蛋白抗原的相对分子质量大小及表达形式。DEAE及PEI凝胶纯化重组蛋白。Western印迹及酶联免疫吸附测定法（ELISA）分析重组蛋白的免疫原性。结果：构建了具有正确基因序列的重组表达载体，在大肠杆菌Bk。（DE3）中诱导表达，重组蛋白占菌体总蛋白的40％，Westem印迹结果证实该重组蛋白能与抗结核分支杆菌多克隆抗体发生特异免疫结合反应。ELISA分析中，该纯化的重组抗原能区别抗结核抗体阳性患者血清及正常人血清。结论：实验中获得了能高效表达16000蛋白抗原的大肠杆菌工程菌，以可溶性形式存于胞浆内。该重组蛋白具有特异的免疫原性。结核病仍为危害全球健康的主要疾病之一，为了改进目前诊断试剂的特异性和研究筛选有价值的亚单位基因工程菌苗，近年来人们对其致病菌的基因及相关蛋白质进行更深入的研究，以期寻找到有应用价值的特异性蛋白质或相关基因，有目的地利用基因工程技术获得重组蛋白抗原为以上研究提供极大的便利。使用结核分支杆菌单克隆抗体TB68筛选出来的16000蛋白抗原，为结核分支杆菌主要的膜蛋白，对其天然提取物的研究结果表明，该蛋白不仅携带有结核分支杆菌特异性的B细胞表位，并能诱导细胞介导的免疫应答反应，具有潜在的诊断应用价值。大量获得重组16000蛋白抗原就为更深入研究该蛋白的生物学、免疫学活性，开发其应用价值提供便利。     

MK—EGFP融合蛋白的构建和表达

目的克隆中期因子（midkine，MK）的编码序列，构建MK—EGFP融合表达质粒，诱导表达并亲合纯化。方法用RT—PCR技术从人胚胎组织中获得MK编码基因，构建原核表达质粒pET—MK-EGFP；转化大肠杆菌，诱导表达重组蛋白；纯化回收后鉴定生物活性。结果重组质粒经酶切测序与预期相符，融合蛋白表达后的指示荧光于激光共聚焦显微镜下清晰可见，MK在其中保持了原有的完整构象和生物活性。结论MK—EGFP重组质粒构建正确，融合蛋白大量表达，并具有完整的生物学活性。     

血小板胶原受体糖蛋白Ⅵ体外表达及功能研究

为了深入研究血小板胶原受体糖蛋白Ⅵ(glycoproteinⅥ，GPⅥ)功能及筛选特异性抑制剂，利用基因重组技术体外表达GPⅥ胞外区片段。采用PCR方法扩增GPⅥ胞外区片段eDNA，构建表达载体pET-20b( )-GPⅥ，转化大肠杆菌BL21(DE3)pLysS，经IPTG诱导表达。表达产物经Ni—NTA Resin纯化、复性：使用Western blot方法鉴定重组蛋白性质；采用胶原结合试验测定重组蛋白的胶原结合能力。结果表明，经测序证明PCR扩增产物与GPⅥ胞外区eDNA序列完全一致；酶切分析证明成功构建了表达载体pET-20b( )-GPⅥ；原核细胞经诱导表达后出现新的相对分子量32kD蛋白条带，诱导产物以包涵体形式存在；Western blot分析表明重组蛋白可与抗Penta—His抗体和抗GPⅥ多抗特异性结合；胶原结合试验表明重组蛋白拥有较好的生物学功能。结论：正确构建了GPⅥ表达载体并成功表达重组蛋白，纯化、复性后的重组蛋白具有良好的抗原性和生物学活性。     

Mutations in the E9L polymerase gene of cidofovir-resistant vaccinia virus strain WR are associated with the drug resistance phenotype

Cidofovir (CDV) is an effective drug against viruses of the Orthopoxviridae family and is active in vitro against variola virus, the cause of smallpox. However, CDV-resistant poxviruses can be generated by repeated in vitro passage in the presence of suboptimal concentrations of CDV. To determine if mutations in the E9L polymerase gene could confer resistance to this nucleoside analog, this gene was sequenced from CDV-resistant vaccinia virus and found to encode five amino acid changes, centered on an N-terminal region associated with 3'-->5' exonuclease activity. Transfer of this mutant E9L gene into wild-type vaccinia virus by marker rescue sufficed to confer the resistance phenotype. E9L polymerase mutations occurred sequentially during passage in CDV, and an H296Y/S338F double mutant that conferred an intermediate CDV resistance phenotype was identified. In vitro, the marker-rescued CDV-resistant vaccinia virus containing all five mutations grew nearly as well as wild-type vaccinia virus. However, the virulence of this virus for mice was reduced, as 10- to 30-fold more CDV-resistant virus than wild-type virus was required for lethality following intranasal challenge. Cidofovir and hexadecyloxypropyl-cidofovir gave partial protection to mice infected with the virus when used at 50 and 100 mg/kg of body weight given as single treatments 24 h after virus exposure, whereas 2-amino-7-[(1,3-dihydroxy-2-propoxy)methyl]purine (compound S2242) was completely protective at 25, 50, and 100 mg/kg/day when given daily for 5 days. These findings suggest that drug therapy for poxviruses may be complicated by drug resistance but that treatment of the infection with currently known compounds is possible.     

Development of a 1-step cell-based assay for cost-effective screening of antiviral drugs for vaccinia virus

We report a 1-step assay to screen antiviral substances combining the simultaneous application of cells, virus sample, and drug to 96-well plates. The results were obtained within 26 h when vaccinia virus plaques were counted or virus-induced cytopathic effect was measured. This fast cost-effective procedure may be used for other cytopathic viruses and is suitable to 1st screening tests.     

MicroRNA Regulation of Glycoprotein B5R in Oncolytic Vaccinia Virus Reduces Viral Pathogenicity Without Impairing Its Antitumor Efficacy

Vaccinia virus, once widely used for smallpox vaccine, has recently been engineered and used as an oncolytic virus for cancer virotherapy. Their replication has been restricted to tumors by disrupting viral genes and complementing them with products that are found specifically in tumor cells. Here, we show that microRNA (miRNA) regulation also enables tumor-specific viral replication by altering the expression of a targeted viral gene. Since the deletion of viral glycoprotein B5R not only decreases viral pathogenicity but also impairs the oncolytic activity of vaccinia virus, we used miRNA-based gene regulation to suppress B5R expression through let-7a, a miRNA that is downregulated in many tumors. The expression of B5R and the replication of miRNA-regulated vaccinia virus (MRVV) with target sequences complementary to let-7a in the 3′-untranslated region (UTR) of the B5R gene depended on the endogenous expression level of let-7a in the infected cells. Intratumoral administration of MRVV in mice with human cancer xenografts that expressed low levels of let-7a resulted in tumor-specific viral replication and significant tumor regression without side effects, which were observed in the control virus. These results demonstrate that miRNA-based gene regulation is a potentially novel and versatile platform for engineering vaccinia viruses for cancer virotherapy.     

Oncolytic vaccinia virus for the treatment of cancer

INTRODUCTION: Gene therapy offers promising approaches for the development of anticancer agents with new modes of action. Among gene therapy vectors, vaccinia virus has emerged as an attractive agent especially when used as an oncolytic virus. AREAS COVERED: This review describes the use of vaccinia virus in cancer therapy as a gene therapy vector, as an oncolytic virus and in the generation of oncolysates. The main achievements of each field are summarized with a special emphasis on vaccinia as an oncolytic vector and its combination therapies. The virus that has advanced furthest in clinical trials, GM-CSF expressing JX-594, is described in detail and its preclinical and clinical data are reviewed. EXPERT OPINION: Vaccinia virus has great potential in cancer gene therapy, especially when used as an oncolytic virus. In particular, JX-594 has shown promising preclinical and clinical data, and a multi-continental randomized Phase III trial in hepatocellular carcinoma is expected to start soon.     

Oncolytic vaccinia virus for the treatment of cancer

Introduction: Gene therapy offers promising approaches for the development of anticancer agents with new modes of action. Among gene therapy vectors, vaccinia virus has emerged as an attractive agent especially when used as an oncolytic virus.

Areas covered: This review describes the use of vaccinia virus in cancer therapy as a gene therapy vector, as an oncolytic virus and in the generation of oncolysates. The main achievements of each field are summarized with a special emphasis on vaccinia as an oncolytic vector and its combination therapies. The virus that has advanced furthest in clinical trials, GM-CSF expressing JX-594, is described in detail and its preclinical and clinical data are reviewed.

Expert opinion: Vaccinia virus has great potential in cancer gene therapy, especially when used as an oncolytic virus. In particular, JX-594 has shown promising preclinical and clinical data, and a multi-continental randomized Phase III trial in hepatocellular carcinoma is expected to start soon.     

Target-organ treatment of neurotropic virus diseases: efficacy as a chemotherapy tool and comparison of activity of adenine arabinoside, cytosine arabinoside, idoxuridine, and trifluorothymidine

A sensitive in vivo system was studied for use in evaluating relatively small quantities of antiviral compounds. Swiss mice were injected intracerebrally with 10 LD(50) of type 1 herpes simplex virus (HSV) or vaccinia virus. Test compounds were injected intracerebrally into mice at a standard time interval after virus inoculation. Significant increases in the number of survivors in drug-treated, infected animals as compared to saline-treated virus controls was the criterion for evaluation of antiviral activity. 9-beta-d-Arabinofuranosyladenine (Ara-A) was used to determine an optimal time of therapy for this system. Significant survival increases occurred when the drug was injected 3, 6, 12, and 48 hr after HSV inoculation, with maximal effect occurring after the 6-hr treatment interval. Determination of brain virus titer of 6-hr Ara-A- and saline-treated mice at various intervals indicated that treatment with the drug delayed development of detectable levels of HSV by 1 day. 1-beta-d-Arabinofuranosylcytosine (Ara-C), trifluorothymidine (TFT), and 5-iodo-2'-deoxyuridine (IDU) were also evaluated with the use of the 6-hr treatment time against HSV and vaccinia virus. Ara-A and TFT increased the number of survivors among mice infected with either virus, whereas Ara-C increased the survivors among HSV-infected, but not vaccinia virus-infected, mice. IDU was inactive against both viruses.     

A Novel Target and Approach for Identifying Antivirals against Molluscum Contagiosum Virus

The dermatological disease molluscum contagiosum (MC) presents as lesions restricted solely to the skin. The poxvirus molluscum contagiosum virus (MCV) is responsible for this skin disease that is easily transmitted through casual contact among all populations, with greater frequency in children and immunosuppressed individuals. In addition, sexual transmission of MCV in adolescents and adults is a health concern. Although the skin lesions ultimately resolve in immunocompetent individuals, they can persist for extended periods, be painful, and result in scarring. Treatment is problematic, and there is no drug that specifically targets MCV. The inability of MCV to propagate in cell culture has impeded drug development. To overcome these barriers, we integrated three new developments. First, we identified a new MCV drug target (mD4) that is essential for processive DNA synthesis in vitro. Second, we discovered a small chemical compound that binds to mD4 and prevents DNA synthesis in vitro. Third, and most significant, we engineered a hybrid vaccinia virus (mD4-VV) in which the natural vaccinia D4 (vD4) gene is replaced by the mD4 target gene. This hybrid virus is dependent on mD4 for viral growth in culture and is inhibited by the small compound. This target system provides, for the first time, a platform and approach for the discovery and evaluation of new therapeutics that can be used to treat MC.     

Simple and inexpensive fluorescence-based technique for high-throughput antimalarial drug screening

Radioisotopic assays involve expense, multistep protocols, equipment, and radioactivity safety requirements which are problematic in high-throughput drug testing. This study reports an alternative, simple, robust, inexpensive, one-step fluorescence assay for use in antimalarial drug screening. Parasite growth is determined by using SYBR Green I, a dye with marked fluorescence enhancement upon contact with Plasmodium DNA. A side-by-side comparison of this fluorescence assay and a standard radioisotopic method was performed by testing known antimalarial agents against Plasmodium falciparum strain D6. Both assay methods were used to determine the effective concentration of drug that resulted in a 50% reduction in the observed counts (EC(50)) after 48 h of parasite growth in the presence of each drug. The EC(50)s of chloroquine, quinine, mefloquine, artemisinin, and 3,6-bis-epsilon-(N,N-diethylamino)-amyloxyxanthone were similar or identical by both techniques. The results obtained with this new fluorescence assay suggest that it may be an ideal method for high-throughput antimalarial drug screening.     

Rapid detection and differentiation of human pathogenic orthopox viruses by a fluorescence resonance energy transfer real-time PCR assay

BACKGROUND: The orthopox viruses that are pathogenic for humans include variola major virus (VAR), monkeypox virus (MPV), cowpox virus (CPV), and to a lesser extent, camelpox virus (CML) and vaccinia virus (VAC). PCR is a powerful tool to detect and differentiate orthopox viruses, and real-time PCR has the further advantages of rapid turnaround time, low risk of contamination, capability of strain differentiation, and use of multiplexed probes. METHODS: We used real-time PCR with fluorescence resonance energy transfer technology to simultaneously detect and differentiate VAR, MPV, CPV/VAC, and CML. An internal control generated by cloning and mutating the PCR target gene facilitated monitoring of PCR inhibition in each individual test reaction. RESULTS: Strain differentiation results showed little interassay variability (CV, 0.4-0.6%), and the test was 100-fold more sensitive than virus culture on Vero cells. Low copy numbers of DNA could be detected with > or =95% probability (235-849 genome copies/mL of plasma). CONCLUSIONS: The real-time PCR assay can detect and differentiate human pathogenic orthopox viruses. The use of an internal control qualifies the assay for high sample throughput, as is likely to be needed in situations of suspected acts of biological terrorism, e.g., use of VAR.     

Mechanism of inhibition of vaccinia virus DNA polymerase by cidofovir diphosphate

Cidofovir (CDV) is a broad-spectrum antiviral agent that has been approved for clinical use in the treatment of cytomegalovirus retinitis. It has also been used off label to treat a variety of other viral infections, including those caused by orf and molluscum contagiosum poxviruses. Because it is a dCMP analog, CDV is thought to act by inhibiting viral DNA polymerases. However, the details of the inhibitory mechanism are not well established and nothing is known about the mechanism by which the drug inhibits poxvirus DNA polymerases. To address this concern, we have studied the effect of the active intracellular metabolite of CDV, CDV diphosphate (CDVpp), on reactions catalyzed by vaccinia virus DNA polymerase. Using different primer-template pairs and purified vaccinia virus polymerase, we observed that CDV is incorporated into the growing DNA strand opposite template G's but the enzyme exhibits a lower catalytic efficiency compared with dCTP. CDV-terminated primers are also good substrates for the next deoxynucleoside monophosphate addition step, but these CDV + 1 reaction products are poor substrates for further rounds of synthesis. We also noted that although CDV can be excised from the primer 3' terminus by the 3'-to-5' proofreading exonuclease activity of vaccinia virus polymerase, DNAs bearing CDV as the penultimate 3' residue are completely resistant to exonuclease attack. These results show that vaccinia virus DNA polymerase can use CDVpp as a dCTP analog, albeit one that slows the rate of primer extension. By inhibiting the activity of the proofreading exonuclease, the misincorporation of CDV could also promote error-prone DNA synthesis during poxvirus replication.