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双语推荐：腺病毒疫苗

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目的 构建含有SIVgag基因的DNA疫苗和重组腺病毒疫苗,为后期在SIV感染的猴模型中进行多载体疫苗联合免疫策略的治疗效果评价奠定基础.方法 将SIV gag基因按照哺乳动物偏嗜密码子进行优化并构建至pVR载体,作为DNA疫苗.以Western Blot方法比较优化前后gag基因表达水平.将优化后的gag基因插入重组腺病毒载体,构建rAd5-SIVgag疫苗.在BALB/c小鼠中分别比较DNA疫苗及rAd5-SIV.gag疫苗单独及联合免疫的效果.结果 密码子优化的SIVgag基因的表达水平远高于野生型SIVgag基因.重组腺病毒疫苗免疫一次或两次诱导的细胞免疫反水平分别高于DNA疫苗免疫一次或两次.两种疫苗联合免疫的反应水平与腺病毒疫苗免疫两次的水平相当,高于DNA疫苗单独免疫及腺病毒疫苗单独免疫一次的结果.结论 成功优化了SIV gag基因,使其不依赖Rev高水平表达；成功构建了表达优化后SIV gag基因的DNA疫苗和rAd5疫苗,可以在小鼠体内诱导较强的gag基因特异性CTL应答.
Objective To construct DNA and recombinant adenovirus vector vaccines containing SIV gag gene for future evaluation of therapeutic effects of combined immunization strategy in SIV-infected macaque models.Methods The modified SIV gag gene was cloned into pVR vector to get DNA vaccine.The wild type and codon-modified SIV gag gene expression was analyzed by Western Blot assay.Then rAdSSIVgag vaccine expressing modified SIV gag was constructed using AdmaxTM system.BALB/c mice were immunized with DNA or rAd5 vaccine once or twice alone or in DNA prime/rAd5 boost strategy.Gag specific cellular immune responses were detected by IFN-γ ELISPOT assay two weeks post the last immunization.Results The codon modification increased the expression of Gag protein significantly.The cellular immune responses in mice immunized with rAd5 vaccine alone once or twice were higher than that of DNA vaccine.And the immune responses elicited by DNA prime/rAd5 boost were comparative with that of immunization with

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目的 构建1b型丙型肝炎病毒(HCV) NS34b基因重组腺相关病毒载体并了解其在HEK 293细胞中的表达,为进一步研究HCV重组腺相关病毒疫苗及其树突状细胞疫苗奠定前期基础.方法 收集基因1b型的丙型肝炎病人血清,用RT-PCR的方法扩增NS3-4b全长片段,与腺相关病毒的表达载体pAAV.CMV.eGFP重组,构建pAAV.CMV.HCV.NS3-4b重组表达载体,转染HEK293细胞,检测其蛋白表达情况.结果 PCR扩增获得的NS3-4b条带与预期大小(2838 bp)一致,重组质粒经双酶切和测序证实NS3-4b基因已重组成功,重组质粒转染HEK 293细胞后Western Blot图可见有目的蛋白表达.结论 成功构建腺相关病毒重组HCV NS3-4b载体并且其能在真核细胞中表达.
Objective To clone 1b type of HCV NS3-4b Gene and express in HEK 293 cells,lay the foundation for further study of the HCV NS3-4b recombinant adeno-associated virus vaccine and its dendritic cell vaccine.Methods HCV 1b patients'' serum was collected,and full length NS3-4b segment was amplified by RT-PCR and cloned into adeno-associated virus'' expression vector pAAV.CMV.EGFP in order to express in HEK 293 cells.At last,it was validated whether express or not by Western Blot.Results The 1b type gene NS3-4b were amplified and consistent to the expected size(2838 bp),the recombinant plasmid has been confirmed its successful restructured by double enzyme and sequencing,at last,Western Blot map can see objective protein expression after it transfect HEK 293 cells.Conclusion The adenoassocisted virus recombination HCV NS3-4b plasmid have successfully constructed and it can express in eukaryotic cells.

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对不同细胞进行牛血清中牛源病毒检测的比较实验，选出用于牛血清中病毒检测的最适合外源病毒增殖的细胞，从而提高血清中病毒的检出率，以确保用于疫苗生产的牛血清安全可靠。将牛腹泻病毒、牛腺病毒、牛细小病毒、牛副流感病毒、呼肠孤病毒和狂犬病毒接种到培养好的MDBK细胞、VERO细胞、BT细胞中，逐日观察细胞的病变情况，待观察到10％细胞出现病变或者培养7 d后，进行免疫荧光染色，选出适合用于六种病毒增殖的细胞系。结果显示，BT细胞对所有病毒敏感，VERO细胞对BAV、BVDV病毒不敏感，MDBK对REO、BVDV、RV三种病毒不敏感。综合比较后，选择BT细胞为BPIV、BPV、BAV、BVDV的增殖及实验用细胞，VERO细胞为REO、RV的增殖及实验用细胞。
Choose the most suitable cell that was used in testing for exogenous virus in bovine serum, then as sensitive cells for detecting the virus in bovine serum in order to improve the detection rate of virus in the serum, it ensured that the bovine serum which was used in production of vaccine was safe. Be ready to cultivate appropriate cells including MDBK, VERO, BT. At the same time,six kinds of virus referred to the BPV, BVDV, BAV, BPIV, REO, RV, Six kinds of virus was inoculated on these three cells. Observing the cells which was inoculated virus every day. After 10% cell was appeared lesion or the cell was cultivated for 7 days, take out the cell, and these cells were dyeing by immunofluorescence, then according to the staining results to determine which cell was suitable for detecting these virus. The results showed that BT cell was sensitive to all cells, Vero cell was not sensitive to BAV, BVDV, MDBK was not sensitive to REO, BVDV, RV. The results of this study showed tha

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目的探讨E型沙眼衣原体（Ct）主要外膜蛋白（MOMP）DNA疫苗和重组腺病毒联合免疫小鼠诱导的免疫效应。方法构建、纯化重组腺病毒Ad-MOMP及重组真核表达质粒pVAX1-MOMP。设计4种免疫方案，分别为DNA免疫（ DNA组）、重组腺病毒免疫（ Ad组）、DNA初次免疫-重组腺病毒加强免疫（ DNA/Ad组）、重组腺病毒初次免疫-DNA加强免疫（ Ad/DNA组）。末次免疫后2周检测小鼠血清特异IgG、IgG1、IgG2a、IgA抗体，阴道分泌物SIgA抗体及脾淋巴细胞分泌IFN-γ、IL-10水平。结果DNA组诱导较弱免疫应答，未产生SIgA抗体及Th1反应。 Ad组诱导出Th1反应及SIgA抗体，且血清抗体显著高于DNA组。联合免疫均能诱导明显强于单独免疫的黏膜SIgA、血清抗体及Th1反应。 Ad/DNA组的Th1反应强于DNA/Ad组；而DNA/Ad组的血清抗体和黏膜抗体水平强于Ad/DNA组。结论Ad-MOMP能诱导黏膜免疫及Th1细胞免疫应答，DNA/Ad及Ad/DNA联合免疫产生的特异性免疫应答明显强于单独免疫。其中Ad/DNA的Th1反应优势更明显，DNA/Ad的血清抗体和黏膜抗体反应更强。接种顺序会影响联合免疫的强弱及类型，这为Ct疫苗的设计研究提供新的思路和实验依据。
Objective To investigate the specific immune responses induced by DNA vaccine com-bined with recombinant adenovirus carrying major outer membrane protein ( MOMP) gene of Chlamydia trachom-atis (Ct) serovar E.Methods Recombinant eukaryotic expression plasmid pVAX 1-MOMP and recombinant adenovirus Ad-MOMP were constructed and purified .Four immunization strategies were designed including DNA immunization (DNA group), recombinant adenovirus immunization (Ad group), DNA prime-recombinant ade-novirus boost regimen ( DNA/Ad group ) and recombinant adenovirus prime-DNA boost regimen ( Ad/DNA group) .Two weeks after the final immunization , vaginal wash specimens , blood samples and spleens were col-lected from all mice for the evaluation of humoral and cellular immune responses .Results Th1 responses and mucosal responses were not found in DNA group .Ad group induced both Th 1 responses and local mucosal re-sponses , and its IgG and IgA levels were significantly higher than those in DNA group

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通过研究重组白细胞介素-2(IL-2)对口蹄疫病毒(FMDV)多表位疫苗免疫效果的增强作用,为IL-2作为FMDV表位疫苗佐剂应用提供实验依据。6周龄雌性Balb/c小鼠,分为4组,第1组联合免疫表达猪IL-2的重组腺病毒(rAd5poIL-2)和串联表达FMDV多表位基因的重组腺病毒(rAd5EGS),第2、3和4组分别注射rAd5EGS、灭活疫苗和PBS,间隔2周免疫1次,共免疫3次。首免后每周采血分离小鼠血清,通过ELISA检测血清中特异性IgG;首免后6周检测血清中抗体亚型IgG1、IgG2a及细胞因子IL-4和IFN-γ的表达水平,同时分离脾细胞,MTT法检测淋巴细胞增殖指数。结果显示,联合免疫rAd5poIL-2和rAd5EGS既能增强rAd5EGS诱导小鼠特异性IgG、IgG1和IgG2a的分泌,又能增强其诱导淋巴细胞增殖能力,且免疫效果强于灭活疫苗;细胞因子检测结果显示,联合免疫rAd5poIL-2后能增强rAd5EGS诱导小鼠IL-4和IFN-γ的分泌,且其诱导IL-4和IFN-γ分泌能力也强于灭活疫苗。说明重组IL-2能有效增强FMDV多表位疫苗诱导抗体分泌能力和细胞免疫应答,rAd5poIL-2有望作为FMDV表位疫苗的候选佐剂。
To investigate the recombinant interleukin-2 (IL-2)enhancing the immune effect of foot-and-mouse disease(FMD)multi-epitope vaccine in mice,to provide the experimental evidence for studying ap-plication of IL-2 as an adjuvant for FMD epitope vaccine,6 weeks old female Balb/c mice were divided into 4 groups,mice of group 1 were combined immunized with recombinant adenovirus expressing porcine IL-2 (rAd5poIL-2)and recombinant adenovirus expressing FMDV multi-epitopes (rAd5EGS),mice of group2,3 and 4 were injected with rAd5EGS,inactivated vaccine and PBS respectively;injection once every two week intervals for 3 times.The sera from mice were collected every week after immunization and the specific IgG was detected by ELISA;the secretion levels of IgG1,IgG2a,IL-4 and IFN-γ in serum were measured by ELISA after 6 weeks;Spleen cells were isolated after 6 weeks and lymphocyte proliferation index was measured by MTT assay.The results of ELISA and MTT showed that combined immunization

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目的生殖器疱疹尚无彻底治愈的方法。研制有效的生殖器疱疹病毒疫苗是预防和治疗HSV-2感染的关键,文中探讨制备腺病毒介导的HSV-2 g D基因修饰的树突状细胞(dendritic cell,DC)疫苗的可行性。方法将HSV-2 g D蛋白基因克隆到质粒载体Shuttle-2,重组质粒酶切鉴定、测序鉴定后构建重组腺病毒p Adeno-HSV-2 g D。分离小鼠骨髓DC细胞,p Adeno-HSV-2 g D转染DC细胞后用流式细胞术检测DC表型,用免疫组化法、RT-PCR、SDS-PAGE和Western blot方法检测HSV-2 g D的表达情况。结果以HSV-2病毒DNA为模板,采用g D基因引物扩增出相应的目的片段。琼脂糖凝胶电泳显示,PCR产物g D基因同预期的大小一致(1182 bp)。p Adeno-g D DNA用脂质体法转染293细胞10 d,反复冻融获得p AdenoHSV-2 g D重组腺病毒,活性为4×1010IU/m L。流式细胞仪检测结果显示:成熟DC和腺病毒感染后DC,CD40含量为(74.2±3.9)%、(81.3±3.1)%,CD80含量为(73.9±4.1)%、(80.4±2.9)%,CD86含量为(76.1±5.5)%、(83.7±3.9)%,均较不成熟DC的CD40、CD80、CD86含量[(9.7±0.5)%、(7.5±1.2)%、(5.2±1.1)%]升高(P0.01)。p Adeno-HSV-2 g D-DC和细胞因子刺激诱导成熟的DC细胞表面分子表达差异无统计学意义(P0.05)。RT-PCR、免疫组织化学方法证明HSV-2 g D能在DC细胞内表达,表达产物的SDS-PAGE和Western blot分析发现,在相对分子质量为43000处有外源蛋白表达,与预期蛋白条带一致。结论成功制备了p Adeno-HSV-2 g D-DC疫苗。
Objective Up to now, there has been no sure cure for genital herpes (GH), and vaccine seems a most promis-ing approach to the prevention and treatment of herpes simplex virus Ⅱ(HSV-2) infection.In this study, we investigated the feasibili-ty of preparing a dendritic cell ( DC) vaccine modified by the adenovirus-mediated HSV-2 gD gene. Methods We subcloned the HSV-2 gD gene into the vector Shuttle-2 and constructed the recombinant adenovirus pAdeno-HSV-2 gD following identification by en-zyme digestion and DNA sequence analysis .We isolated DCs from the mouse bone marrow , analyzed their phenotypes by flow cytome-try after transfection with the recombinant adenovirus pAdeno-HSV-2 gD, and determined the expression of HSV-2 gD by immunohisto-chemistry, RT-PCR, SDS-PAGE, and Western blot. Results Based on HSV-2 DNA, the corresponding target fragments were am-plified with the gD gene primers.Agarose gel electrophoresis showed the correct size of the PCR product (1182 bp) as predi

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为研究马立克病毒强毒感染与疫苗免疫鸡组织病理学变化的差异,试验采用108只1日龄海兰褐公雏鸡,随机分为对照组(C组)、马立克病毒CVI988疫苗株腹腔注射免疫组(IA组)和京-1株攻毒组(V组)。在接种后3、6、10、15、20、26、35、45、60d,对3组各取4只鸡进行剖检,分别取肺脏、胸腺、骨骼肌、肾脏、睾丸、肝脏、心肌、腺胃、脾脏、法氏囊、坐骨神经等组织于100mL/L中性福尔马林中固定,石蜡包埋,制备切片,HE染色,光镜下进行组织病理学观察。结果与对照组相比,V组在接种6d时,无明显病理变化,IA组淋巴器官内出现淋巴细胞增多;接种10d后,V组脾脏动脉周围淋巴鞘、淋巴小结内淋巴细胞数量减少;15d后胸腺小体消失,出现肿瘤细胞,其他组织均出现增生性团块,有大小不等的肿瘤细胞;IA组可见脾脏中淋巴细胞增多,动脉周围淋巴鞘脾小结增多,胸腺小体中淋巴细胞增多,其他组织有明显的淋巴细胞浸润。由此可见,鸡感染马立克病毒后免疫器官损伤,各组织器官都出现明显病理学变化;鸡接种疫苗后免疫器官淋巴细胞逐渐增多,但以后也出现免疫器官损伤与其他组织器官淋巴细胞增多,由此表明了CVI988疫苗的免疫毒性,为研究无免疫毒性且有较好免疫力的马立克病疫苗提供理论依据。
This study was designed to investigate the influence of Gallid herpesvirus 2 Marek''s Disease chal-lenge virus and atteunated vaccine virus CVI 988/Rispens on changes of histopathology in chickens .108 one day old chicks were randomly divided into a control group (C) ,an experimental group challenged with Jing-1 challenge strain (V) ,and a third experimental group injected intra-abdominally (IA) with vaccine strain ,CVI988/Rispens .Different organs of lung ,thymus ,skeletal muscle ,kidney ,testis ,liver ,cardiac muscle ,stomachus glandularis ,spleen ,bursa of Fabricius ,sciatic nerve were removed immediately from 4 chicks of every group sacrificed by cervical dislocation on days 3 ,6 ,10 ,15 ,20 ,26 ,35 ,45 and 60 ,and were fixed overnight in 100 mL/L neutral formalin ,and embedded in paraffin wax for routine histological procedures .Paraffin sections were stained with hematoxylin and eosin . The histopathological changes were evaluated with light microscope .It was found tha

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目的 评价HPV16 E6E7的复制缺陷型重组5型腺病毒(PK-HPV-ad5)治疗性疫苗对实验小鼠免疫应答和抗肿瘤的生物学效应.方法 使用基因重组技术构建PK-HPV-ad5疫苗,并通过小鼠免疫试验,检测小鼠总抗体和特异性IFNγ,同时将造模小鼠分成疫苗组和对照组,分别对其进行抑瘤试验、TC-1肿瘤细胞挑战试验和肿瘤切除后防复发试验.结果 HPV16 E6E7诱导的总抗体第12天的水平相对较高(1:400～1:600);3批次疫苗特异性IFNγ在第14天与对照组比较分别升高8.6、5.9和8.9倍,差异有统计学意义(t=15.721、6.967和14.342,P均＜0.01).抑瘤试验表明疫苗剂量为107IU/只时小鼠肿瘤生长率为0,与对照组比较差异有统计学意义(确切概率法,P＜0.01),3批次疫苗验证有效剂量为107IU/只时肿瘤抑制率可达80％(8/10)以上.TC-1肿瘤细胞挑战试验结果显示:小鼠先接种疫苗能引起特异性的免疫应答,并能保护90％(9/10)的小鼠免受TC-1肿瘤细胞的攻击;肿瘤切除后防止复发试验提示在注射相同剂量疫苗时,对104个/只和105个/只肿瘤细胞造模小鼠,第0、5天免疫组肿瘤复发数少于第5,8天免疫组(1/10,4/10 vs 8/10,7/10).结论 PK-HPV-ad5疫苗能诱导小鼠产生特异性的免疫应答,对抗肿瘤复发有治疗潜力.
Objective To evaluate the immune responses and anti-tumor effects of replication-deficient recombinant adenovirus-5 vector vaccine of human papillomavirus type 16 E6E7 as a theraputic vaccine (PK-HPV-ad5) in mouse models.Methods PK-HPV-ad5 vaccine was constructed by gene recombination technique.HPV16E6E7 total antibody and specific IFNγ of the vaccine were detected by mouse immune experiment.The model mice were divided into vaccine group and control group,and were used for anti-tumor test,TC-1 tumor cell challenge test and evaluation of tumor excision combined with vaccine to prevent tumor recurrence.Results HPV16 E6E7 total antibody increased to a comparatively high level (1:400-1:600) on d12 after vaccination.The specific IFNγin 3 batches of vaccine(20121201,20121202 and 20121203) were 8.6,5.9 and 8.9 times of those in control group on d14 after vaccination,respectively,with significant differences(t=15.721,6.967 and 14.342,P all＜0.01).The anti-tumor test showed that the

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[目的]采用家蚕丝腺生物反应器这一蛋白高效表达系统，尝试建立一种高效、低廉、稳定生产狂犬病病毒核蛋白（RVNP）的技术体系，为生产基因工程疫苗奠定基础。[方法]通过RT-PCR方法克隆获得RVNP序列，采用家蚕丝胶蛋白基因（Ser1）启动子为特异性启动子，将RVNP构建到含有增强型绿色荧光蛋白（EGFP）筛选标记基因的piggyBac转座子表达载体（pBA3EGFP）中，并采用显微注射转基因家蚕技术构建家蚕丝腺生物反应器。[结果]G1代通过EGFP标记的筛选获得转基因家蚕阳性蛾区26个，蛾圈阳性率为59.09%。PCR实验证实RVNP已经整合到家蚕基因组中，Western blot 分析表明 RVNP 蛋白可能附着于丝胶蛋白并随家蚕吐丝行为分泌到蚕茧中。[结论]获得了能够表达外源的RVNP蛋白的转基因家蚕品系，该系统有望成为高效、低廉、稳定地生产狂犬病基因工程疫苗的技术体系之一。
[Objective]In order to obtain a potent technology which can produce high value RVNP protein and then lay a foundation for the production of vaccines, the silk gland bioreactor was employed as an expression system in this study.[Method]The nucleoprotein gene of rabies virus ERA strain (RVNP) was cloned by RT-PCR, and the RVNP was firstly connected to the downstream of middle silk gland-specific Ser1 promoter, and then inserted into pBA3EGFP, an expression vector which contained an EGFP reporter gene. By using the transgenic method, the silk gland bioreactor of piggyback-mediated transgenic silkworm was obtained. [Result]Twenty-six broods of transgenic silkworms (G1) were obtained and the GFP-positive percentage of broods was 59.09%. Further PCR identification indicated that RVNP had been integrated into silkworm genome, and Western blot analysis showed that the protein might be attached to the sericin which was secreted into the cocoon with the spinning of transgenic silkworms. [Conclus

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目的：通过构建在六邻体中嵌合表达乙肝病毒表面抗原preS1上两个中和抗原表位的人3型重组腺病毒，鉴定应用此策略所展示表位的抗原性。方法利用overlap PCR技术扩增出在HVR1和HVR2中分别引入乙肝病毒表面抗原中和表位KR359和KR127的嵌合六邻体基因，酶切后连接入穿梭质粒pBR322-L/R，然后与骨架质粒pBRAdΔE3GFP共转化至BJ5183中重组，得到阳性克隆pBRAdΔE3GFP-preS1。将其线性化后转染293细胞拯救病毒，再大量扩增并纯化。同时应用表达载体pGEX-4T3表达融合蛋白GST-KR359KR127，将重组病毒和GST融合蛋白分别免疫BALB/c小鼠获得多抗血清。 ELISA、Western blot实验鉴定嵌合表位的抗原特性。结果人3型腺病毒载体成功的在衣壳六邻体表面展示了乙肝病毒表面抗原中和表位KR359和KR127，并且诱导产生的多抗血清能识别GST融合表达抗原以及天然的乙肝病毒表面抗原。结论衣壳融合策略置换高变区来展示外源中和表位的方法可行，并且可以通过同时置换多个高变区来达到加强免疫效果或者多价抗原免疫保护的作用，为人3型腺病毒六邻体嵌合载体系统在疫苗研究中的应用奠定基础，同时也为新型乙肝疫苗的研制奠定基础。
Objective To construct a hexon-chimeric human adenovirus type 3 ( HAd3 ) vector expressing two neutralizing epitopes of hepatitis B surface antigen preS 1 (HBsAg-preS1) and to analyze the antigenicity of the chimeric epitopes .Methods Two neutralizing epitopes of HBsAg-preS1 including KR359 and KR127 were inserted into hypervariable region 1 ( HVR1) and hypervariable region 2 ( HVR2) of HAd3 hexon .Chimeric hexon gene encoding the two epitopes was amplified by overlap PCR and then subcloned in -to shuttle plasmid pBR322-L/R containing the homologous recombination regions .The digested shuttle plas-mid containing chimeric hexon gene was co-transfected into Escherichia coli BJ5183 cells together with back-bone plasmid pBRAdΔE3GFP to construct pBRAdΔE3GFP-preS1 vector.Then pBRAdΔE3GFP-preS1 vector was digested with AsiSⅠand transfected into AD293 cells to construct recombinant virus (rAD3E-preS1). CsCl gradient centrifugation was used for purification .Glutathione S-transfer

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