荧光定量PCR用于重组杆状病毒鉴定及病毒滴度检测的研究

目的：建立一种高效、简便的荧光实时定量PCR方法,用于重组杆状病毒鉴定及病毒滴度的检测.方法：利用Bac-to-Bac载体系统在E.coli菌株DH10 Bac中构建重组杆状病毒穿梭质粒（Bacmid）和在昆虫细胞中构建含人IL-18基因的重组杆状病毒,纯化的重组Bacmid作为PCR检测的标准模板,由昆虫细胞中收获的病毒母液用于空斑测定和病毒DNA提取.以10倍梯度稀释的重组Bacmid作为标准模板,进行荧光定量PCR扩增IL18基因片段并绘制标准曲线,然后以提取的重组杆状病毒DNA作为模板,采用同样体系进行实时PCR反应检测.同时,以琼脂糖空斑法测定病毒母液的滴度.结果：成功构建了重组杆状病毒并建立了病毒滴度的实时荧光PCR检测方法.运用标准模板进行的PCR反应显示该方法的线形范围为101～108拷贝,病毒母液的DNA拷贝浓度（vg/ml）值约为空斑检测的滴度pfu/ml值的10倍.结论：荧光定量PCR方法可灵敏快速地鉴定重组杆状病毒,并在较大的线性范围内检测重组杆状病毒滴度,较之空斑法更准确地反映了重组杆状病毒的实际数量.     

绿色荧光标记重组人偏肺病毒空斑形成滴度测定方法的建立

目的 建立用于绿色荧光标记的重组人偏肺病毒(GFP-rhMPV)空斑形成滴度测定方法.方法 基因组中插入绿色荧光蛋白基因的hMPV全序列cDNA质粒和主要蛋白质表达质粒转入细胞293T后获得感染性重组hMPV.GFP-rhMPV在Vero-E6细胞中连续传代提升病毒滴度并保存.将等倍稀释的重组病毒液接种常规制备的Vero-E6细胞单层,用含或不含胰酶的低熔点琼脂精凝胶覆盖细胞,孵育一定时间后,采用荧光显微镜下计数荧光空斑数和抗原抗体蓝斑形成法计算病毒滴度.结果 感染后3 d,荧光显微镜下GFP-rhMPV可在低熔点琼脂糖凝胶覆盖层下形成分界较为清晰的绿色荧光集落,接种后3 d荧光空斑相对独立,便于计数.蓝斑形成法在感染后第5天蓝斑较大,易观察.此前拯救获得的GFP-rhMPV在宿主细胞Vero-E6中的复制滴度可达1×106 PFU以上.结论 成功建立了GFP-rhMPV的空斑形成实验滴度定量检测方法,为hMPV的致病机制、防治手段研究奠定了基础.     

人TNF-αmRNA实时荧光定量PCR检测标准的构建

目的：克隆人TNF-α cDNA，作为人TNF-α mRNA定量检测的标准品。方法：用RT-PCR法从人外周血单个核细胞的总mRNA中逆转录扩增TNF-α的cDNA，将纯化的TNF-α cDNA与pUCm-T载体进行连接，转化宿主菌DH-5α，然后提取重组质粒DNA，用限制性核酸内切酶Hind Ⅲ,EcoR Ⅰ进行双酶切鉴定并测序分析，最后对质粒标准进行实时荧光定量PCR检测。用聚乙二醇沉淀法纯化质粒并检测λ200nm吸光度，确定原液的重组质粒拷贝浓度并以此制备荧光定量PCR梯度浓度标准品。结果：酶切鉴定、PCR扩增及测序分析均证实TNF-αcDNA重组到pUCm—T载体上。结论：成功克隆了TNF-α实时荧光PCR定量标准。     

马尔堡、埃博拉病毒双重荧光定量PCR检测方法的建立

目的 建立一种快速、敏感、特异的双重实时荧光定量PCR方法,可同时检测马尔堡病毒和埃博拉病毒.方法 通过序列比对挑选出两种病毒基因组中高度保守的序列,分别设计引物及Taqman探针,两条探针分别标记FAM和Texas Red荧光报告基因,建立双重实时荧光定量PCR反应体系.结果 双重荧光定量PCR方法检测两种病毒阳性标准品的灵敏度分别为30.5拷贝/μl和28.6拷贝/μl,通过检测日本脑炎病毒、黄热病毒、登革热病毒无交叉反应,有较好的灵敏度和特异性.结论 建立了马尔堡、埃博拉病毒双重荧光定量PCR检测方法,实现了两种病毒同时实时定量检测,在传染病防控领域有较好的应用前景.     

Hsa-mir-196b慢病毒表达载体的构建及鉴定

目的构建Hsa-mir-196b慢病毒表达载体并对其进行鉴定。方法以正常人外周血DNA为模板,PCR扩增得到目的基因。通过HpaⅠ/XhoⅠ双酶切及其后的连接将其插入Lentilox3.7（pLL-3.7）质粒中。PCR筛选阳性克隆,测序鉴定。用pLL-3.7-mir-196b、pCMV-VSV-G和pCMV-dR8.91三质粒系统共感染HEK-293FT细胞,包装生产慢病毒。将所得病毒悬液梯度稀释后感染HEK293FT细胞,以检测病毒滴度,并用实时定量PCR检测慢病毒感染后Hsa-mir-196b的表达变化。结果 PCR及测序结果证明成功构建了pLL-3.7-mir-196b重组质粒。所得慢病毒上清滴度为（7.2±1.1）×107TU/ml。感染慢病毒的239FT细胞中,Hsa-mir-196b的表达量相对于未感染细胞提高了约24倍。结论成功构建Hsa-mir-196b慢病毒表达载体。     

狂犬病病毒实时荧光定量PCR检测方法的建立和阳性对照的制备

目的 建立狂犬病病毒实时荧光定量PCR检测方法,制备狂犬病病毒(rabies virus,RV)的假病毒颗粒阳性对照.方法 在RV的N基因保守区设计引物和探针,建立反转录实时荧光定量PCR检测方法,克隆得到噬菌体MS2的装配蛋白和壳蛋白基因(MS2),将其重组到质粒pET-28b(+)中,再将RV的N基因片段重组到MS2下游,经原核表达得到RV假病毒颗粒.结果 实时荧光定量PCR检测RV重组质粒最低检测限为15拷贝/μl,重组表达质粒经原核表达可形成耐RNase的RV病毒样颗粒.结论 建立了反转录实时荧光定量PCR检测RV核酸的方法,成功构建了可耐受RNase且无传染性的RV阳性对照.     

小鼠IL-10重组慢病毒载体的构建和病毒包装

目的:构建小鼠IL-10重组慢病毒表达载体并进行包装,为进一步研究IL-10基因修饰树突细胞在哮喘免疫耐受中的作用提供实验基础。方法:小鼠IL-10基因片段经PCR扩增后,与酶切线性化的慢病毒载体进行定向连接,获得PGC-LV-IL-10重组慢病毒并转化细菌感受态细胞。克隆菌落行PCR鉴定,对阳性的重组质粒进行测序并转入293T细胞,荧光显微镜下观察GFP表达并行Western blot鉴定。将重组质粒与慢病毒包装系统一同转染293T细胞进行病毒包装,Real-time定量PCR法检测病毒滴度。结果:DNA测序结果及Western blot鉴定证实成功构建小鼠IL-10重组慢病毒载体,对其进行包装后测定慢病毒滴度为2×108TU/ml。结论:成功构建并包装小鼠IL-10重组慢病毒表达载体。     

重组杆状病毒表达的EIAV Env蛋白与含有env基因重组痘苗病毒的联合免疫

目的：构建新型的马传染性贫血病毒(EIAV)的候选疫苗：方法：利用BAC—To—BAC杆状病毒表达系统，将中国马传贫驴白细胞弱毒疫苗(EIAV DLV)及其亲本株(EIAV LN)env基因导入到杆状病毒基因组中。转染昆虫细胞后，得到的重组病毒用SDS—PAGE和Western blot检测表达产物。以本实验室构建的含有EIAV env基因的重组痘苗病毒，单独或与重组杆状病毒表达的EIAV Env蛋白联合免疫小鼠。结果：构建的重组杆状病毒能正确表达全长Env蛋白。与单独免疫组相比，联合免疫组免疫应答显著增强，其中中和抗体的滴度提高5～9倍。结论：含有EIAV env基因的重组痘苗病毒与Env蛋白抗原联合免疫，能够诱导高滴度的中和抗体。     

荧光定量PCR法在重组逆转录病毒pLXSN-BDNF滴度测定中的应用

目的制备含大鼠脑源性神经营养因子（BDNF）基因的逆转录病毒，建立荧光定量PCR测定滴度的方法。方法扩增BDNF基因，构建重组质粒pLXSN-BDNF，将其转染包装细胞，经G418筛选、病毒浓缩后用荧光定量PCR法和克隆形成法测定滴度。结果经PCR、酶切和测序鉴定，BDNF基因成功插入逆转录病毒载体中，筛选得到稳定的分泌重组病毒细胞系。重组病毒100倍浓缩后经荧光定量PCR法和克隆形成法测定的滴度分别为6．92×10^6拷贝/ml和3．2×10^5CFU/ml，两种方法测得的结果差异有统计学意义（P〈0．05）。结论成功扩增BDNF基因，制备了重组病毒pLX—SN-BDNF，建立了荧光定量PCR检测滴度的方法，为基因治疗青光眼性视神经损伤的实验研究提供重要参考指标。     

辛德毕斯病毒荧光PCR检测方法的建立

目的建立辛德毕斯病毒核酸的SYBRGREENΙ荧光PCR检测方法。方法根据辛德毕斯病毒基因组核苷酸序列特性设计引物。病毒在BHK21细胞上繁殖扩增后提取RNA和逆转录。以病毒cDNA为模板分别进行SYBRGREENΙ荧光PCR和常规RTPCR扩增,并对SYBRGREENΙPCR法的灵敏性、特异性、重复性等进行分析。结果最适退火温度为55℃,最适引物浓度为0.5μmol/L。用该方法检测2株SIN病毒株YN87448和XJ160结果均为阳性,而对其他虫媒病毒如甲病毒属Geta病毒、乙脑病毒、Batai病毒、Banna病毒、环状病毒及西方马脑炎病毒合成模板检测时结果均为阴性。根据病毒空斑形成实验结果,将YN87448病毒悬液进行连续10倍稀释后分别用常规PCR和SYBRGREENΙ荧光PCR方法进行检测。结果SYBRGREENΙ荧光PCR检测敏感性比常规PCR方法要高近100倍,检出下限可达0.1PFU/ml。对模拟感染的人血清标本检测结果表明人血清中成分对检测体系无明显影响。对151份不明原因发热和病毒性脑炎患者的血清或脑脊液标本进行检测,结果检测到6份标本阳性。结论本实验建立了辛德毕斯病毒特异性核酸的SYBRGREENΙ荧光PCR检测方法,实验结果显示了较好的特异性、广谱性,初步证实可应用于临床标本的检测,为将来用于临床和调查辛德毕斯病毒在我国的流行情况提供了新的技术手段。     

A rapid method for estimation of baculovirus titer based on viable cell size

Baculovirus protein expression system is a powerful tool for producing recombinant proteins. To optimize conditions for efficient recombinant protein expression, it is important to determine titer of virus stock for arriving at an optimal multiplicity of infection (MOI) that maximizes recombinant protein expression. Traditionally plaque assays have been used for titer determination. Other methods such as endpoint dilution, quantitative real-time polymerase chain reaction and flow cytometry have been developed to aid the determination of virus titers. However, most of these methods are time-consuming and labor intensive. In this regard, a simple and rapid method for determination of virus titers based on the cytopathic effects that lead to viable cell size increase following virus infection is presented in this paper. In this study, the Vi-CELL (Beckman Coulter) was used to measure cell-diameter over a range of virus dilutions, following infection. Applying statistical modeling techniques, the viable cell-diameter data was used to estimate the virus titer. The results indicated that the viable cell-diameter based titer estimation to be reliable and comparable to titers determined by the traditional plaque assay.     

Bac-to-Bac杆状病毒表达系统表达小鼠GITRL蛋白并鉴定

目的:利用Bac-to-Bac杆状病毒表达系统表达小鼠糖皮质激素诱导的肿瘤坏死因子受体的配体(G ITRL)蛋白。方法:用EcoRⅠ和SalⅠ双酶切GITRL-PMD18T载体,回收目的基因G ITRL,并定向克隆入穿梭质粒pFastBacHTA中。以pFastBacHTA-G ITRL转化感受态DH10Bac细菌,在DH10Bac细菌内重组pFastBacHTA与杆粒发生转座。筛选阳性克隆,提取重组杆粒,转染Tn昆虫细胞株,获取完整的重组杆状病毒。反复感染Tn细胞,扩增病毒同时表达目的蛋白,用Western blot法进行蛋白鉴定。结果:经核苷酸序列测定及PCR鉴定,成功地获得含GITRL基因的重组杆粒。在杆粒转染的Tn细胞中表现有细胞病变,推断转染成功并获得重组杆状病毒。Western blot法鉴定显示,在Mr20000处有特异性的蛋白条带。结论:利用Bac-to-Bac杆状病毒表达系统成功地表达了小鼠GITRL蛋白,为进一步研究其生物学活性及功能奠定了实验基础。     

Improved assay to detect neutralizing antibody following vaccination with diluted or undiluted vaccinia (Dryvax) vaccine

The assessment of immunogenicity of a diluted vaccinia vaccine for possible widespread use of a diluted vaccine in the event of a bioterrorist attack prompted us to focus on the development of a sensitive and specific plaque reduction neutralization (PRN) assay to assess the antibody response of volunteers to a vaccinia (Dryvax) vaccine. Two incubation times, 1 h or overnight (approximately 15 h), were explored for the neutralization step of the assay. In addition, serum samples were evaluated using both sonicated and nonsonicated virus in PRN assays with 1 and 15 h of incubation. The use of the overnight incubation method resulted in the detection of antibody in two vaccinated individuals who exhibited a take, i.e., a major reaction indicative of successive vaccination as defined by the Centers for Disease Control and Prevention, but did not have a fourfold increase in antibody to vaccinia virus by the 1-h-incubation method and increased the sensitivity from 94 to 100%. In addition to the increased sensitivity of the assay, we noted a significant increase (approximately 40-fold) in the PRN titer of serum samples tested with the 15-h-incubation method. The use of sonicated virus increased the reproducibility of the virus titers and PRN titers. Forty-two percent of the samples tested using sonicated virus had a PRN titer that was fourfold higher or greater than that of nonsonicated virus in the assay. A PRN titer that was threefold higher or greater was observed in more than half (58%) of the samples using sonicated virus. Therefore, the more sensitive, specific, and reproducible plaque neutralization assay for the detection of antibody to vaccinia virus is the method using a 15-h-incubation time and freshly sonicated vaccinia virus.     

Rhinovirus detection: comparison of real-time and conventional PCR

Rhinoviruses are important human respiratory viruses and the major causative agents of the common cold. Historically, detection of rhinovirus has been by virus culture and this was significantly improved by the use of PCR assays. Recently real-time PCR was developed but to date there have been no reported comparisons of conventional and real-time PCR assays for detection of rhinovirus. In this study, we first compared real-time PCR (SYBR Green I) to conventional PCR for the detection of rhinovirus in serially diluted standard DNA and rhinovirus stock to determine the limits of detection. Next, assays were compared for sensitivity to detect rhinovirus in cell culture with a known number of infected cells. Finally, the assays were compared using clinical samples known to contain rhinovirus. Real-time PCR was 10-fold more sensitive than conventional PCR to detect rhinovirus in standard DNA and in virus stock and >10-fold more sensitive to detect rhinovirus in cultured cells. Real-time PCR was significantly superior for detection of rhinovirus in patients' nasal aspirates (sensitivity 72% versus 39%, P < 0.05). In summary, we found that real-time PCR was more sensitive than conventional PCR and reduced post-PCR processing. Hence, real-time PCR is suitable for both research and clinical purposes.     

构建含肿瘤坏死因子相关凋亡诱导配体基因慢病毒载体

背景：腺病毒的表达时间有限,会限制目的基因的表达时间和表达量,不利于实验的持续进行,故文章选择慢病毒作为载体,与目的基因大鼠肿瘤坏死因子相关凋亡诱导配体基因片段进行基因重组。 目的：探讨构建含有大鼠肿瘤坏死因子相关凋亡诱导配体基因慢病毒载体的方法。 方法：根据GenBank中大鼠肿瘤坏死因子相关凋亡诱导配体序列(NM 145681.1),设计出该基因的特异性引物Trail-AgeⅠ-F和Trail-AgeⅠ-R,用PCR法从大鼠cDNA文库中扩增出目的基因,AgeⅠ酶切将该基因克隆入真核表达载体GV218中,产生目的重组质粒,用脂质体 Lipofeetamine 2000包裹构建的重组质粒和辅助包装载体,3个质粒共同转染293T细胞,产生含有表达肿瘤坏死因子相关凋亡诱导配体蛋白的慢病毒颗粒,收集病毒进行滴度鉴定,收集细胞提取蛋白进行基因表达情况的检测。 结果与结论：筛选出的克隆阳性菌测序获得肿瘤坏死因子相关凋亡诱导配体基因,全长861 bp,与GenBank中发表的肿瘤坏死因子相关凋亡诱导配体核苷酸序列完全一致。包装后的病毒LV-mTrail在转染2 d后收集病毒液,经PCR和Western blot方法鉴定,从基因和蛋白的层面均证实携带有目的基因肿瘤坏死因子相关凋亡诱导配体基因。孔稀释法及实时荧光定量PCR病毒滴度测定法测定结果为2×109 TU/mL。提示大肠杆菌同源重组法能有效和方便地构建出含有肿瘤坏死因子相关凋亡诱导配体基因的慢病毒载体。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

High Expression of Insulin-like Growth Factor H (IGF-Ⅱ) Using Bac-to-Bac Expression System

Objective In order to obtain mature insulin-like growth factor- Ⅱ ( IGF- Ⅱ ), we used Bac-to-Bac baculovirus expression system. Methods Firstly the IGF- Ⅱ cDNA was cloned into a donor plasmid pFastBac1 and the recombinant pFastBac1 was then introduced into competent cells DH 10Bac. Recombinant bacmids were constructed by transposing a mini-Tn7 element from a donor plasmid pFastBac1 to the mini-attTn7 attachment site on the bacmid where the Tn7 transposition functions were provided in trans by a helper plasmid, and then used to transfect Sf9 insect cells to get recombinant baculovirus. The recombinant baculovirus was used to infect insect cells. Results Agarose gel analysis showed that recombinant donor plasmid pFastBac1 was constructed successfully; Agarose gel analysis of PCR products confirmed recombinant bacmid ; SDS-PAGE and Western Blotting showed that a 7KD protein band appeared. Conclusion The mature IGF- Ⅱ with immunogenecity has been expressed and produced by using Bac-to-Bac expression system.     

A genotype-independent real-time PCR assay for quantification of hepatitis B virus DNA

Accurate quantification of hepatitis B virus (HBV) DNA levels is important for monitoring patients with chronic HBV infection and for assessing their responses to antiviral therapy. This study aimed to develop a real-time PCR assay that is sensitive and can accurately quantify a wide range of HBV DNA levels across the known HBV genotypes. An "in-house" real-time PCR assay using primers and a TaqMan probe in a highly conserved region of the HBV surface gene was designed. The assay was standardized against a WHO standard and validated against plasmids of HBV genotypes A through H. The linear quantification range was approximately 5 x 10(0) to 2.0 x 10(9) IU/ml. Results of samples from patients infected with HBV genotypes A through H tested using our real-time "in-house" PCR assay showed an excellent correlation with those of the Cobas Amplicor HBV Monitor (R2=0.9435) and the Cobas TaqMan HBV (R2=0.9873) tests. We have established a real-time PCR assay that is genotype independent and can accurately quantify a wide range of HBV DNA levels. Further studies of additional samples are ongoing to validate the genotype independence of our assay.