转基因番茄防龋疫苗中外源目的基因鉴定及表达检测

背景:转基因植物可食性疫苗是以植物作为载体,将外源性基因整合到植物基因组当中,进一步激活动物或人体免疫系统以获得特异性免疫能力。但外源性基因的持续低表达量一直无法达到满意的免疫效果。目的:用分子生物学技术检测转基因番茄植株中pacA-ctxB融合基因表达及目的蛋白的表达量,为进一步观察可食用防龋疫苗的防龋效果提供研究基础。方法:(1)提取转基因番茄叶片总DNA,PCR检测外源性融合基因pacA-ctxB,实验分组3组:阳性对照组为质粒p2355-EPC10;空白对照组为普通番茄1株(红抗219);转基因组为转基因番茄9株;(2)BCA法检测转基因番茄果肉中总蛋白的水平;Western blot证实转基因番茄果实中PAcA/CTB目的蛋白的表达,目的蛋白检测分组2组:转基因组为表达外源性嵌合基因的转基因番茄5株(PCR检测阳性);空白对照组为普通番茄1株;酶联免疫吸附实验(ELISA)法检测转基因番茄果实中PAcA/CTB目的蛋白浓度。结果与结论:(1)PCR扩增结果可见9株转基因番茄中有5株出现约1.7 kb特异性扩增条带,占总检测植株的55%;(2)转基因番茄总蛋白为3.15 g/L...     

HCMV pp150抗原基因在蚕豆中的遗传转化及鉴定

目的研制人巨细胞病毒（HCMV）口服疫苗。方法根据HCMV基质磷蛋白PP150基因序列设计引物，PCR法从HCMV AD169株基因组DNA中扩增出编码PP150抗原决定簇区域的基因片段。将Kozak序列插入pp150基因的起始密码子的上游，3＇端引入了内质网滞留信号KDEL序列，将修饰后的基因片段克隆进载体pBI121，构建了带潮霉素（Hyg）选择抗性基因的植物表达载体pCAMBIA1300／pp150和根癌农杆菌工程菌EHA105；采用叶盘法转化蚕豆，获得了5株抗性植株，通过PCR、Southern blot和RNA dot blot分析鉴定，确认了3株为转基因植株。通过ELISA和Western blot对这3株的蛋白萃取物进行分析，以鉴定其免疫学活性。结果这3株转基因植株表达的PP150蛋白具有免疫原活性。结论这些转基因植株为HCMV口服疫苗的研究提供了条件。     

人源轮状病毒vp7基因的克隆与转基因植物研究

目的：克隆人源轮状病毒外壳蛋白vp7基因，以制备转基因植物疫苗。方法：用RT-PCR方法制备vp7基因，以植物高效表达质粒PBI121为载体，构建重组DNA质粒。重组体用载体上的通用引物为测序引物，鉴定克隆的正确性。再将鉴定过的重组质粒用农杆菌介导法转染马铃薯外植体，分别用PCR、Western blot法鉴定阳性转化植株中vp7基因的表达。结果：成功地将vp7转入马铃薯植株中，并且在转化植株中检测出了vp7的表达。结论：成功地构建了重组PBI121/hvp7质粒，获得表达外源基因vp7的转基因马铃薯。     

酸性成纤维细胞生长因子植物表达载体的构建及转化烟草的初步研究

为了降低aFGF的生产成本,结合植物生物反应器的优点,就aFGF在转基因烟草中的表达进行了探索。将aFGF基因插入植物表达载体pBI121中,获得了含有aFGF基因的植物表达载体pBI121-TΩAB-aF,利用农杆菌介导法将基因转化烟草,转基因烟草在含有卡那霉素和头孢霉素的培养基培养,获得具有卡那抗性的转基因烟草植株,通过PCR检测、RT-PCR检测及Western blot检测证实外源基因已经在烟草中成功表达。为植物生物反应器生产aFGF提供了理论基础。     

鼠源轮状病毒vp7基因的表达载体构建及转基因植物的研究

目的：构建表达鼠源轮状病毒抗原基因vp7的植物表达载体及植物转化研究。方法：抗原基因vp7经PCR扩增后直接克隆到PUC－T载体，双酶切目的片断和植物表达载体，以T4连接酶将目的片断与载体相连接，电转化方式将重组质粒转入农杆菌。结果：以重组农杆菌为介导将靶基因转入到马铃薯外植体。成功地将目的基因定向克隆到表达载体，并转入到农杆菌中；以农杆菌为介导获得抗性转基因马铃薯植株。结论：通过PCR检测，初步确定轮状病毒外壳蛋白基因vp7已成功地整合到马铃薯植株中。     

抗HTNV mAb 3G1单链抗体基因转化植物的研究

目的 构建抗HTNV mAb 3G1 scFv的转基因拟南芥植株.方法 从含有3G1 scFv基因的重组质粒中酶切获得含有该目的基因的表达框,将其克隆入载体pCAMBIA2301,构建植物表达载体3G1scFv-pCAMBIA2301.通过农杆菌介导的花粉管法将其转入拟南芥,PCR和Southern blotting检测是否获得转基因植株.组织化学染色检测标记基因GUS是否表达.结果 限制性内切酶酶切鉴定结果证明3G1 scFv基因被成功克隆入植物表达载体pCAMBIA2301,构建获得3G1scFv-pCAMBIA2301重组质粒.PCR和Southern blotting检测证明获得抗HTNV mAb 3G1 scFv的转基因拟南芥植株.组织化学染色结果表明,标记基因亦高效表达.结论 成功地将外源基因转入拟南芥中并表达,为进一步研究利用植物表达医用抗体奠定了基础.     

A组轮状病毒结构蛋白基因在马铃薯细胞中的初步表达

目的 在马铃薯细胞中表达轮状病毒结构蛋白。方法采用RT-PCR扩增截短VP4蛋白基因，克隆于植物表达载体paBI221，再双酶切pBI221，将带有CaMV35S启动子、外源基因及终止子的片段转入pSB11，通过三亲杂交方法，制备农杆菌转化载体pSB111-VP4，并对pSB111-VP4作点杂交检测，筛选鉴定重组子，对马铃薯组织细胞进行转化。结果转化细胞经Western印迹检测其免疫活性，具有良好的抗原性。结论为研制轮状病毒重组疫苗、亚单组疫苗、转基因植物口服疫苗做了前期准备。     

轮状病毒抗原基因在马铃薯细胞中的表达和活性检测

目的 轮状病毒引起的婴幼儿腹泻病，在发展中国家有较高的发病率和病死率，因此，研制安全有效的轮状病毒疫苗成为当务之急。方法 本文采用RT－PCR扩增截短VP4蛋白基因，克隆于植物表达载体pBI221,再双酶切pBI221,将带有CaMV35S启动子、外源基因及终止子的片段转入pSB11,通过三亲杂交方法，制备农杆菌转化载体pSB111-VP4,并对pSB111-VP4作为杂交检测，筛选鉴定重组子，并对马铃薯组织细胞进行转化。结果 Western印迹检测其免疫活性，且具有良好的抗原性。结论 该转化系统的建立，为研制轮状病毒重组疫苗、亚单组疫苗、转基因植物口服疫苗做了前期准备。     

抗汉滩病毒中和性鼠/人嵌合抗体基因转化拟南芥的初步研究

目的:建立能表达抗汉滩病毒中和性鼠/人嵌合抗体基因的转基因植株,为汉坦病毒基因工程抗体的研究提供实验基础.方法:将构建的含抗汉滩病毒中和性鼠/人嵌合抗体基因的植物抗体表达质粒3G1MH-pCAMBIA2301,用TSS冻融法导入农杆菌GV3101,利用农杆菌介导的抽真空转基因技术转化野生型拟南芥,获得转基因植株.用PCR、Northern blot检测转基因植株.结果:经PCR分析表明,拟南芥的基因组中有抗汉滩病毒中和性鼠/人嵌合抗体基因整合,并成功获得7株T0代转基因拟南芥.植株提取RNA,经Northern blot分析可见约1 500 bp及800 bp处的目的条带,为编码重、轻链的目的基因.结论:成功地构建了含抗汉滩病毒中和性鼠/人嵌合抗体基因的拟南芥植株,为进一步利用植物表达治疗性抗体奠定了基础.     

重组人β-防御素-2基因的植物表达载体的建立

目的许多证据表明利用转基因植物作为生物反应器规模化生产高附加值的药用重组蛋白质,其技术可行、成本低廉、效益显著,具有广阔的应用开发前景.本研究拟运用植物转基因技术,尝试构建高效表达hBD-2的植物生物反应器的可能性.方法将C端带Myc和6xHis双标记的hBD-2的重组DNA克隆到植物真核表达载体pCAMBLA1303中,构建C端带Myc和6xHis双标签的hBD-2的重组植物真核表达载体;并将此载体转化根癌农杆菌LBA4404,利用卡那霉素进行抗性基因筛选,确定根癌农杆菌的阳性克隆;通过根癌农杆菌将外源基因转入马铃薯植物愈伤组织细胞,利用潮霉素抗性基因筛选及PCR检测,监测hBD-2在其中的遗传表达.结果(1)酶切分析、PCR验证和DNA测序等证据表明,C端6个组氨酸的hBD-2已被正确地插入pCAMBIA1304载体中CaMV35S和Nos终止子之间,成功构建了重组hBD-2/His基因的植物表达载体rpCAMBIA1304/hBD-2/His.(2)rpCAMBIA1304/hBD-2/His已成功地转化根癌农杆菌LBA4404,阳性克隆菌株已获得.(3)潮霉素抗性进行筛选及PCR检测结果提示hBD-2cDNA基因已整合到马铃薯植物愈伤组织的基因组中.结论结果表明,重组hBD-2/His基因的植物表达载体通过根癌农杆菌介导进入了马铃薯细胞植物愈伤组织.利用植物转基因技术,构建高效表达hBD-2的植物生物反应器具有可能性.     

改进探针标记法斑点杂交在轮状病毒VP7分型中的应用

目的建立一种更加简便的A组人轮状病毒（HRV）核酸斑点杂交VP7分型方法,以便用于对HRV流行情况进行调查。方法在HRVVP7编码基因各G基因型问高度变异而型内高度保守区域设计分型探针,在该区域的两侧相对保守区域设计一对通用引物,利用PCR分别将地高辛标记HRV5种常见型别（G1～4,G9型）的DNA探针,建立基于VP7的斑点杂交方法;选取经抗原检测和聚丙烯酰胺凝胶电泳（PAGE）检测均为HRV阳性的2006至2008年住院腹泻患儿粪便标本200份,RT—PCR扩增VP7全基因,并对扩增阳性产物应用斑点杂交方法进行G型别分析。结果建立的斑点杂交方法在5种型别探针间无交叉反应,各型探针的检测灵敏度可达到10Pg。200份PAGE阳性标本中162份RT—RCR扩增VP7基因阳性,斑点杂交显示G1型41例（25．3％）,G2型2例（1．2％）,G3型63例（38．9％）,G9型35例（21．6％）,混合感染19例（11．7％）,杂交未分出型2例（1．2％）,未检测到G4型HRV。结论本研究所建立的斑点杂交方法敏感度和特异度强,适合在HRV大规模分子流行病学调查时应用。通过该方法的初步应用,发现北京地区婴幼儿HRV除了常见的G1、G2和G3型外,还有G9型感染。     

人轮状病毒G2和G3型主要中和抗原VP7基因在重组腺病毒中的表达

目的 研究我国G2和G3型轮状病毒主要中和抗原VP7基因在重组腺病毒中的表达。方法 在前期成功表达Gl型VP7的基础上，选用我国G2和G3型主要流行株97S43和97S48 VP7基因，用非复制型腺病毒载体对上述基因进行表达。结果 获得了表达我国G2型和G3型人轮状病毒流行株VP7基因的非复制型重组腺病毒rvAdG2VP7和rvAdG3VP7，应用PCR及Southem blot技术证实在重组腺病毒中整合有轮状病毒G2型VP7和G3型VP7基因，RT-PCR证明重组腺病毒在感染的293细胞内均能有效地转录插入基因，Western blot检测到轮状病毒VP7基因的表达。结论 这一工作为进一步进行动物实验，发展多价轮状病毒疫苗打下了基础。     

WU多瘤病毒衣壳蛋白原核表达及初步分析

目的 获得WU多瘤病毒重组衣壳蛋白,分析其抗原性,筛选具有诊断价值的抗原.方法 采用PCR技术扩增WU多瘤病毒衣壳蛋白VP1、VP2和VP3的基因片段,并将目的基因插入PGEX-20T表达载体,转化大肠杆菌BL21(DE3)后诱导表达.用免疫印迹法对表达蛋白进行鉴定和分析并用临床样本进行初步验证.结果 重组质粒在BL21(DE3)菌株中经IPTG诱导、SDS-PAGE和免疫印迹鉴定显示在相对分子质量(Mr) 69×103、63×103和56×103处出现清晰条带,重组质粒经双酶切鉴定,表达产物经GST标签鉴定均正确.16份呼吸道分泌物WU多瘤病毒核酸阳性血清标本和70份阴性标本免疫印迹分析表明,重组蛋白具有良好的抗原性.结论 成功构建了WU多瘤病毒衣壳蛋白VP1、VP2和VP3的表达载体,表达的重组蛋白具有良好的抗原性,为进一步研究提供了资料.

Abstract：

Objective To express the capsid proteins of WU polyomavirus(WUPyV) for research and find antigen for diagnostic value. Methods Coding sequences of capsid proteins of WU polyomavirus by PCR were cloned in prokaryotic expression vector PGEX-20T. Recombinant plasmids were transformed into E. coli BL21(DE3) and induced by IPTG for proteins expression. Recombinant proteins were identified by Western blot. Results SDS-PAGE proved that recombinant proteins showed three bands with molecular relative mass of 69×103, 63×103 and 56×103. The recombinant proteins were recognized by anti-GST McAb. The antigenicity was tested by Western blot using 16 WU polyomavirus positive and 70 negative sera. Conclusion Recombinant VP1, VP2 and VP3 expressed in E. coli can combine with WUPyV-Ab and have good antigenicity. They can be used for further research.     

Detection of human parvovirus B19 DNA by using the polymerase chain reaction.

The polymerase chain reaction (PCR) was investigated for detecting human parvovirus B19 (B19) DNA in sera. Three pairs of oligonucleotides were evaluated as primers. The best oligonucleotide pair spanned 699 nucleotides, including the region common to VP1 and VP2. After PCR amplification of B19 DNA in serum, a 699-nucleotide DNA fragment was detected on agarose gels. This DNA fragment was B19 DNA, because after Southern transfer it hybridized to a 19-nucleotide internal probe and contained a single PstI cleavage site. Dot blot hybridization with a radiolabeled cloned portion of the B19 genome as a probe was compared with PCR. PCR was 10(4) times more sensitive than dot blot hybridization and, with an internal radiolabeled probe, 10(7) times more sensitive than dot blot hybridization. Of 29 serum specimens from 18 patients with proven B19 infections, 24 were PCR positive. None of 20 serum samples from uninfected controls were positive. Of 22 serum samples positive for immunoglobulin M to B19, PCR detected B19 DNA in 17. Seven serum samples lacking immunoglobulin M were PCR positive. PCR detected B19 DNA in urine, amniotic fluid, pleural fluid, ascites, and leukocyte extracts. PCR is a rapid and simple method for diagnosing infections with human parvovirus B19 but must be combined with serologic tests for immunoglobulin M to B19, especially when testing only a single serum sample.     

Pathogen-derived resistance using a viral nucleocapsid gene confers only partial non-durable protection in peanut against peanut bud necrosis virus

Genetic engineering of peanut (Arachis hypogaea L.) using the gene encoding for the nucleocapsid protein (N gene) of peanut bud necrosis virus (PBNV; genus Tospovirus, family Bunyaviridae) was used to impart resistance to bud necrosis disease in peanut (PBND), a disease for which no durable resistance is available in the existing germplasm. Over 200 transgenic lines of peanut var. JL 24 were developed for which integration and expression of the transgenes was confirmed by PCR, Southern hybridization, RT-PCR and western blot analysis. The T₁ and T₂ generation transgenic plants were assayed through virus challenge in the greenhouse by using mechanical sap inoculation at 1:100 and 1:50 dilutions of PBNV, and they showed varying levels of disease incidence and intensity. Greenhouse and field evaluation with T₂ generation plants indicated somewhat superior performance of the three transgenic events that showed considerable reduction in disease incidence. However, only one of these events showed over 75 % reduction in disease incidence when compared to the untransformed control, indicating partial and non-durable resistance to PBND using the viral N-gene.     

Detection of serum hepatitis B virus DNA using the polymerase chain reaction assay

We compared the sensitivity of the polymerase chain reaction (PCR) assay to that of slot blot hybridization for detecting hepatitis B virus (HBV) DNA in the serum of a chimpanzee infected with HBV and 52 patients. Also, we utilized a rapid PCR procedure for the detection: Viral DNA was released from virions by incubating serum with NaOH. After a primary PCR amplification, the sample was reamplified using a second set of primer pairs (PCR/PCR). In the chimpanzee, HBV DNA was detected 3 weeks earlier than the appearance of hepatitis surface antigen (HBsAg) and persisted for two weeks with antibody to HBsAg. Of the 14 chronic hepatitis B patients positive for both HBsAg and HBV e antigen (eAg), 9 were positive for HBV DNA by slot blot hybridization and all 14 by PCR. Also, of 9 patients positive for HBsAg and antibody to eAg, 2 were positive for HBV DNA by slot blot hybridization and 8 by PCR. Three of the 11 patients who had lost HBsAg during follow up examination of chronic hepatitis B were positive for HBV DNA by PCR, whereas none of them was positive by slot blot hybridization. Six patients who had recovered from acute hepatitis B more than one year ago and 12 cases who had had vaccination of HBV were negative for HBV DNA by PCR. This technique should yield valuable information on the biology of HBV.