Development of an efficient recombinant mosquito densovirus-mediated RNA interference system and its preliminary application in mosquito control

The Aedes aegypti densovirus (AeDNV) has potential as a delivery vector for foreign nucleic acids into mosquito cells. In this study, we investigated the ability of plasmids containing recombinant viral transducing genome to induce RNA interference (RNAi) effects in C6/C36 cells. We then evaluated the efficiency of a recombinant AeDNV vector to induce RNAi in Aedes albopictus larvae. We found that the expression of V-ATPase was inhibited by up to 90% at 96 h post-transfection in transfected C6/C36 cells. In addition, the bioinsecticidal activities of various RNAi-expressing AeDNV vectors used to infect Ae. albopictus larvae were also tested. We found that when Ae. albopictus larvae were infected with recombinant AeDNV, expression of V-ATPase was downregulated by nearly 70% compared to controls. Furthermore, the median survival time bioassays demonstrated that recombinant AeDNV caused more serious pathogenic effects than the wild type virus. This is the first report showing that recombinant virus plasmid and corresponding recombinant AeDNV can be used as an effective in vitro and in vivo RNAi delivery system, respectively.     

博尔纳病病毒pEGFP-p24基因重组质粒的构建及鉴定

构建博尔纳病病毒pEGFP-p24基因重组表达质粒。通过PCR方法扩增获得博尔纳病痛毒p24基因的完整序列,将此片段定向克隆到pEGFP-N1载体多克隆位点区,筛选重组阳性菌株,提取重组质粒,利用PCR方法和核酸序列测定验证重组质粒构建的正确性。PCR及核酸序列测定证明博尔纳病病毒pEGFP-p24基因重组表达质粒构建成功。构建的重组质粒将为研究博尔纳病病毒p24基因在真核细胞中的功能和作用提供实验依据。     

The densovirus of Periplaneta fuliginosa (PfDNV) as an insect vector for persistent foreign gene expression in vivo

An infectious clone of the Periplaneta fuliginosa densovirus (PfDNV) has been constructed and the PfDNV genome can rescue from the plasmid and replicate as the wild-type virus in nymphs of P. fuliginosa. To investigate the ability of the cloned PfDNV genome to be used as a stable and persistent expression vector, we constructed seven recombinant plasmids in which the GFP reporter gene was inserted into the genome of PfDNV. When these recombinant constructs were transfected into hosts, the GFP was expressed efficiently in every clone. Southern blot analysis revealed that recombinant plasmids had integrated into host genome. Infectious recombinant virions could be produced from plasmids in which the GFP gene was downstream of and in frame with the NS3 and NS1 coding regions. These results indicate that PfDNV genome can be used as an insect vector for the transfer and persistent expression of an exogenous gene.     

鹅源新城疫病毒NP、P和L基因的克隆与P基因的表达鉴定

将鹅源新城疫病毒的NP、P和L基因通过RT-PCR方法从尿囊液中扩增后分别克隆进pGEM—T easy载体,再分别亚克隆到真核表达载体pCI—neo上,通过酶切、PCR和测序验证克隆正确。利用P基因开放性阅读框(ORF)上靠近终止密码上游的AgeI位点,将报告基因绿色荧光蛋白(GFP)基因克隆进P基因真核表达重组质粒,分别转染COS-1细胞和CEF细胞,在倒置荧光显微镜下可见到绿色荧光,表明GFP基因已得到表达,由此证明P基因也已得到表达。鹅源新城疫病毒NP、P和L基因的克隆成功,为即将进行的鹅源新城疫病毒的反向遗传操作以及功能基因组研究打下基础。     

拯救细胞系恢复重组家蚕核型多角体病毒包涵体实现外源蛋白在家蚕中产业化生产

构建家蚕Bombyx mori肌动蛋白（BmA3）启动子驱动的家蚕核型多角体病毒（BmNPV）多角体基因（ph）和OpNPV极早期启动子（IE1）驱动的zeocin抗性筛选基因转座供体载体,与鳞翅目辅助转座质粒pie2piggyBac共转染家蚕卵巢细胞BmN,经200μg/ml zeocin抗生素筛选一个月,成功获得持续表达BmNPV多角体蛋白的稳定细胞系BmN-A3ph。多角体缺陷型重组病毒BmBac-GF P感染拯救细胞系BmN- A3ph, 细胞成功装配出病毒包涵体颗粒,其包装效率约为野生型病毒感染正常BmN细胞的8%。用拯救型包涵体病毒颗粒喂食家蚕幼虫进行复感染,结果表明稳定细胞系所包装的包涵体病毒与野生型病毒一样能够通过口服途径感染宿主,却并不在宿主体内形成包涵体,从而保证外源基因高效表达。拯救型包涵体病毒可望解决传统注射感染效率较低问题,通过喂食感染可促进杆状病毒介导的家蚕生物反应器产业化进程。     

HIV—1核蛋白p24在昆虫细胞中的表达

将完整的ＨＩＶ－１ ｐ２４基因克隆到杆状病毒转移质粒中,使用重组转移质粒与野生型杆状病毒ＤＮＡ共转染Ｓｆ９昆虫细胞,经筛选获得带有编码ｐ２４基因的重组杆状病毒。重组杆状病毒感染Ｓｆ９细胞后在细胞中表达了ＨＩＶ核蛋白ｐ２４。其重组蛋白的分子量为２４ｋＤ。此重组糖蛋白在免疫荧光,免疫印染和酶联免疫实验中都能被人ＨＩＶ－１阳性血清和单克隆抗体所识别。     

家蚕浓核病毒中国株非结构蛋白1(NS1)的表达

利用PCR技术扩增出BmDNV-3 NS1基因,将目的基因与原核表达载体pET-30a进行连接,转化BL21 star菌并在该菌中表达,经Western blot鉴定表达的产物为BmDNV-3 NS1蛋白,纯化NS1蛋白并制备兔多克隆抗体.同时BmDNV-3 NS1基因亚克隆到杆状病毒转移载体pFastBae-HTb-eGFP中,转化BmDH10BAC感受态细胞,提取的重组Bacmid通过脂质体包埋转染家蚕BmN细胞,再以收获的重组病毒感染家蚕幼虫.家蚕BmN细胞和幼虫感染重组病毒2d后均观察到绿色荧光,经SDS-PAGE分析真核表达的产物与预测的NS1-eGFP融合蛋白大小不一致,说明NS1-eGFP融合蛋白被昆虫内源性的蛋白酶降解.降解的产物用NS1蛋白抗体进行Western blot鉴定为BmDNV-3 NS1蛋白.     

人核糖核酸酶抑制因子基因重组腺病毒载体的构建及鉴定

目的构建含有人核糖核酸酶抑制因子（hRI）基因的重组腺病毒载体。方法以含有全长cDNA的pT7-RI为模板,PCR扩增hRI,经T载体克隆后,酶切亚克隆到穿梭质粒pAdTrack—CMV上,在BJ5183细菌内和pAdEasy-1同源重组。筛选阳性克隆,酶切、PCR及测序鉴定,线性化后脂质体法转染293细胞进行包装、扩增。通过观察绿色荧光蛋白（GFP）的表达及PCR扩增目的基因等方法鉴定重组的腺病毒。结果酶切鉴定及PCR结果证明hRI基因重组腺病毒载体构建成功,病毒滴度为1．5×10^10 pfu/ml。结论应用细菌内同源重组法成功构建了含hRI基因的重组腺病毒载体。     

H5N1亚型禽流感病毒NS1基因在昆虫细胞中的表达

将H5N1亚型禽流感病毒（AIV）NS1基因插入到杆状病毒转移载体pFastBac1中,获得重组转移载体pFastBac1- NS1。将pFastBac1- NS1转化到DH10Bac感受态细胞中,筛选到重组转座子rBacmid-NS1。在脂质体转染试剂介导下将rBacmid-NS1转染对数生长期的Sf9昆虫细胞获得重组杆状病毒rBV-NS1。rBV-NS1感染Sf9细胞后,通过SDS-PAGE、Western blot和ELISA分析表明：获得了分子量为26ku的特异性NS1蛋白;并且该蛋白可与H5N1 AIV攻毒鸭的血清发生特异性免疫反应,而不能与H5N1AIV灭活疫苗免疫鸭的血清发生反应。试验结果表明：NS1在Sf9昆虫细胞中获得了高效表达,具有与天然蛋白相似的免疫活性,并可以作为区分免疫及自然感染个体的鉴别诊断抗原。本实验为建立禽流感病毒自然感染家禽与禽流感灭活苗免疫家禽的鉴别诊断方法奠定基础。     

共表达人p53、GM-CSF和B7-1基因的重组腺病毒的构建

 为开展肿瘤的复合基因治疗 ,构建以串联方式携带人野生型p53、GM CSF和B7 1基因的重组腺病毒穿梭质粒pBB 1 0 2 .将pBB 1 0 2与腺病毒包装质粒GT40 50共转染 2 93细胞 ,通过细胞内同源重组获得重组腺病毒BB 1 0 2 .在 2 93细胞中扩增病毒 ,并通过氯化铯密度梯度超速离心纯化病毒 ,获得高滴度和高纯度的病毒 .分别经免疫组织化学分析、ELISA和流式细胞分析 ,检测BB 1 0 2介导的人野生型p53、GM CSF和B7 1基因在喉癌细胞Hep 2中的表达 .结果表明 ,BB 1 0 2能够有效地将其所携带的目的基因导入Hep 2细胞并使其在细胞中高效表达 ,表达高峰期为转染后 2～ 4d ,此后随时间递减 ,可持续 1 0d以上 .     

Rescue and preliminary application of a recombinant newcastle disease virus expressing green fluorescent protein gene

Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain, seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of the NDV ZJI strain. The pNDV/ZJI, with three helper plasmids, pCIneoNP, pCIneoP and pCIneoL, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, an infectious NDV ZJI strain was successfully rescued. Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP. After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells, the recombinant NDV, NDV/ZJIGFP, was rescued. Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF) cells 48h post-infection, indicating that the GFP gene was expressed at a relatively high level. NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route. Four days post-infection, strong green fluorescence could be detected in the kidneys and tracheae, indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.     

Rescue and Preliminary Application of a Recombinant Newcastle Disease Virus Expressing Green Fluorescent Protein Gene

Based on the complete genome sequence of Newcastle disease virus (NDV) ZJI strain, seven pairs of primers were designed to amplify a cDNA fragment for constructing the plasmid pNDV/ZJI, which contained the full-length cDNA of the NDV ZJI strain. The pNDV/ZJI, with three helper plasmids, pCIneoNP, pCIneoP and pCIneoL, were then cotransfected into BSR-T7/5 cells expressing T7 RNA polymerase. After inoculation of the transfected cell culture supernatant into embryonated chicken eggs from specific-pathogen-free (SPF) flock, an infectious NDV ZJI strain was successfully rescued. Green fluorescent protein (GFP) gene was amplified and inserted into the NDV full-length cDNA to generate a GFP-tagged recombinant plasmid pNDV/ZJIGFP. After cotransfection of the resultant plasmid and the three support plasmids into BSR-T7/5 cells, the recombinant NDV, NDV/ZJIGFP, was rescued. Specific green fluorescence was observed in BSR-T7/5 and chicken embryo fibroblast (CEF) cells 48h post-infection, indicating that the GFP gene was expressed at a relatively high level. NDV/ZJIGFP was inoculated into 10-day-old SPF chickens by oculonasal route. Four days post-infection, strong green fluorescence could be detected in the kidneys and tracheae, indicating that the recombinant GFP-tagged NDV could be a very useful tool for analysis of NDV dissemination and pathogenesis.     

人Tim-3基因真核表达载体的构建及表达

目的：为了构建可在真核细胞中高效表达人Tim-3（hTim-3）的真核表达质粒,以便用于hTim-3肿瘤免疫治疗研究。方法：取健康人外周血的单个核细胞,用高保真聚合酶扩增hTim-3基因,先进行hTim-3基因的亚克隆,用Bgl II和SalI限制性内切酶切下带有酶切位点的hTim-3基因,最终构建hTim-3基因的真核表达质粒pEGFP-N1-hTim-3。用脂质体方法转染质粒至肝癌细胞SMMC7721和巨噬细胞U937中。48h后荧光显微镜观察转染细胞绿色荧光表达情况,初步判断转染效率。结果：酶切和测序鉴定证实目的基因hTim-3正确插入到真核表达载体pEGFP—N1中,转染肝癌细胞SMMC7721和巨噬细胞U937后,在荧光显微镜下观察到绿色荧光蛋白的表达。结论：成功构建了可在真核细胞中高效表达hTim-3基因的真核表达质粒pEGFP-N1-hTim-3,为进一步研究hTim-3的肿瘤免疫治疗奠定了基础.     

伪狂犬病病毒上海株（PRV—SH）gE^—／gI^—／GFP^＋缺失株的构建

在构建了含伪狂犬病病毒（Pseudorabies Virus,PRV）上海株gI基因和gE基因克隆鉴定的基础上,采用酶切的方法构建了载体pgEI。然后用限制性内切酶BamHI和BstPI缺失掉gE基因5‘端363bp,同时把绿色荧光蛋白（GFP）基因表达盒插入到缺失部分,并在下游 引入一个多克隆位点,构建了缺失转移载体pgEI-GFP。用DOTAP转染试剂盒将pgEI-GFP转染了感染PRV-SH的BHK-21细胞,待出现80％病变后收获病毒,并以蚀斑法得到纯化的缺失了gE/gI重组病毒株。小鼠试验证实了缺失株的毒力有所下降。     

汉坦病毒H8205株G1P基因片段重组杆状病毒表达载体的构建及表达

将汉坦病毒H8205株G1P基因的保守序列(约1000bp)作为目的基因插入到BactoBac杆状病毒表达系统的pFastBacHTb供体质粒中,利用Tn7转座子同BacmidDNA同源重组,获得了含目的基因片段的重组杆状病毒DNA,并利用其转染Sf9昆虫细胞,72h后收集细胞悬液,再用该悬液侵染Sf9昆虫细胞,48h后收获病毒.采用IFA分析收获的产物,观察到了特异性的荧光,并且采用SDSPAGE和Western印迹也获得了与预期一致的结果.证明感染后的Sf9昆虫细胞所表达的蛋白中含有能与抗汉坦病毒H8205株多克隆抗体特异性结合目的蛋白.研究表明,采用杆状病毒表达系统可以成功表达出汉坦病毒H8205株包膜糖蛋白G1基因片段,为开发适合的以G1P为抗原的汉坦病毒诊断试剂进行了前期的探索.     

Biosynthesis of recombinant human hepatitis B M-HBsAg in silkworm larvae

The middle surface antigen (M-HBsAg) of human hepatitis B virus is virus envelope protein. It's used as a basis for development of vaccine and test-system for detecting of hepatitis B virus. The cDNA of M-HBsAg was inserted into transfer vector pBK273 under the polyhedron promoter with obtaining of recombinant plasmid DNA pBHep-2. As a result of cotransfection pBHep-2 with wild type BmNPV the recombinant baculovirus rBmNPVHep which included the cDNA of M-HBsAg under the polyhedron promoter was obtained. Infection of silkworm larvae Bombyx mori with recombinant virus resulted in expression of foreign gene and accumulation of middle surface antigen of human hepatitis B virus mostly (>90%) in fat bodies of silkworm larvae.     

具有蛋白酶及解旋酶活性的HCV-NS3重组蛋白的纯化及活性分析

为深入探讨HCV-NS3蛋白的酶动力学性质,制备了具有蛋白酶及解旋酶活性的HCVNS3重组蛋白。利用PCR扩增HCV非结构基因NS3,插入pPIC9,测序分析。携带NS3基因的重组质粒(pPIC9-NS3)转化毕氏酵母菌菌株GS115,甲醇诱导表达NS3蛋白。重组蛋白首先采用Hitrap chelating柱进行亲和分离,之后使用Mono S HR柱进一步纯化。对纯化后的NS3重组蛋白的酶活性进行分析,结果表明,获得的重组蛋白分别具有蛋白酶及解旋酶活性。本研究为深入探讨NS3编码酶的功能和开发抗病毒药物创造条件。     

Expression and identification of mutated osteoprotegerin in culture cells and larvae of silkworm

Osteoprotegerin (OPG, or osteoclastogenesis inhibitoryfactor, OCIF) is a soluble member of the tumor-necrosisfactor receptor family discovered in 1997 that can inhibitosteoclastogenesis and prevent bone loss from resorption.Simonet et al. [1] reported tha…     

异种化人肾细胞癌特异性抗原G250真核表达载体的构建及表达

目的：构建含有人肾细胞癌特异性抗原G250（CAⅨ）主要T细胞表位区域、猴和鼠CAⅨ部分片段区域融合基因tG250的真核表达质粒,并在猴肾COS7细胞中表达。方法：通过基因合成和PCR技术构建人、猴和鼠G250区域融合基因tG250,将其插入含有人Igκ链前导信号肽（sig）、人IgG-Fc和糖基磷脂酰肌醇（GPI）锚定信号肽融合基因序列的细胞膜锚定修饰真核表达载体pCI-Fc-GPI中,继而又将酶切后的sig-tG250-Fc-GPI融合基因导入含有细小病毒内部核糖体结合位点（IRES）基因且可以共表达人GM-CSF和B7.1融合基因的真核表达载体pVAX1-IRES-GM/B7中;将构建的重组质粒pVAX1-sig-tG250-Fc-GPI-GM/B7转染COS7细胞,利用流式细胞仪和免疫荧光检测其表达。结果：测序结果表明tG250融合基因序列正确,PCR和酶切鉴定证明已将其连入真核表达载体pVAX1-IRES-GM/B7中;流式细胞仪和免疫荧光的检测结果显示,重组质粒pVAX1-sig-tG250-Fc-GPI-GM/B7在COS7细胞中得到很好的表达。结论：构建了重组质粒pVAX1-sig-t G250-Fc-GPI-GM/B7,且在COS7细胞中有效表达,为以G250为靶点的抗肾细胞癌基因疫苗的构建与功能研究奠定了基础。     

创建可视化的家蚕杆状病毒表达系统表达家蚕二分浓核病毒非结构蛋白NS1

重组杆状病毒感染昆虫细胞是表达外源蛋白常用的一种方法。为有效鉴定转染的细胞中是否产生了重组病毒粒子,对质粒pFastBacI进行改造,构建了极早期基因ie1启动子控制的绿色荧光蛋白egfp基因表达盒,以及多角体基因启动子控制的外源DNA的一个通用型双表达载体;通过酶切、连接的方式,将家蚕二分浓核病毒ns1基因连接到多角体启动子下游;在转座酶的介导下,该供体质粒上部分序列可转座到穿梭载体Bm-Bacmid上,进而构建可同时表达egfp和ns1基因的重组杆粒。将构建的该重组杆粒DNA转染BmN细胞,通过观察可视化的绿色荧光信号,可迅速判定转染后的细胞中重组病毒粒子产生的情况,收集转染后的细胞培养上清,将其感染BmN细胞,对感染4 d后的细胞总蛋白进行Western blotting分析,结果表明能杂交到一条36 kDa大小的特异蛋白,表明NS1蛋白成功获得了表达,进而为深入研究ns1基因的功能奠定了基础。