流感病毒RNA聚合酶亚基PA6片断的克隆及表达

目的将流感病毒RNA聚合酶PA6亚基片段进行亚克隆、表达,获得重组的亚克隆多肽,为进一步研究PA6亚基功能奠定基础。方法以PA亚基cDNA为模板,用PCR方法扩增出PA6片段,应用定向克隆策略将扩增后片段与高效表达载体pQE32重组,阳性克隆重组质粒经酶切及测序鉴定,IPTG诱导各亚克隆系高效表达,优化表达条件。结果PCR法扩增出402bp的片段,酶切及测序鉴定获得正确的重组质粒,在E.coliM15中表达得到相对分子量约为17KD的重组蛋白,获得重组多肽。结论成功地亚克隆及表达了流感病毒RNA聚合酶PA6亚基。     

IQSEC1通过病毒蛋白PB1调控甲型流感病毒的增殖

目的探讨IQSEC1是否通过病毒蛋白PB1调控甲型流感病毒的增殖。方法首先克隆甲型流感病毒[A/Shanghai/02/2013(H7N9)]的8个基因;其次,通过免疫共沉淀检测IQ模体Sec7结构域蛋白1(IQSEC1)与聚合酶PB1(PB1)存在相互作用;此外,通过过表达或者敲低IQSEC1的方法检测IQSEC1对PB1核定位的影响;最后,过表达或者敲低IQSEC1后检测Influenza A virus[A/Shanghai/02/2013(H7N9)]。结果病毒感染条件下,外源IQSEC1和PB1存在相互作用。当过表达IQSEC1时,细胞中IQSEC1的表达量上升,相应的PB1在细胞核中的定位减少;当用敲低IQSEC1时,细胞中IQSEC1的表达量下降,相应的PB1在细胞核中的定位上升。过表达IQSEC1后,甲型流感病毒的增殖水平下降(P0.05)。敲低IQSEC1后,甲型流感病毒的增殖水平上升(P0.05)。结论 IQSEC1通过减少甲型流感病毒蛋白PB1的核定位抑制甲型流感病毒的增殖。     

嗜肺军团菌免疫原蛋白核酸疫苗诱导的小鼠免疫应答及其保护力

目的:研究嗜肺军团菌免疫原蛋白核酸疫苗诱导的小鼠免疫原性以及对LP感染小鼠的保护能力。方法:用嗜肺军团菌免疫原蛋白基因真核表达重组质粒pcDNA3.1-ip作为DNA疫苗免疫BALB/c小鼠,检测免疫小鼠体内抗原特异性抗体水平、脾淋巴细胞增殖活性、IFNγ-产生水平和CTL特异杀伤活性等指标,以评价疫苗的免疫原性。真核表达重组质粒pcDNA3.1-ipDNA疫苗重复免疫BALB/c小鼠2次,末次免疫2周后,用10倍LD50剂量攻击小鼠,计数小鼠的存活数及小鼠肺中的细菌数,观察感染鼠的肺部病理变化。结果:pcDNA3.1-ip免疫小鼠后诱导产生了特异的体液免疫应答和细胞免疫应答,免疫组的免疫原性和免疫保护性均高于对照组pcDNA3.1( )组(P<0.01)。结论:免疫原蛋白基因可作为嗜肺军团菌核酸疫苗的侯选基因。     

新型甲型H1N1流感病毒HA基因DNA疫苗诱导小鼠产生中和抗体

目的 探讨新型甲型流感病毒(2009H1N1)血凝素(HA)DNA疫苗诱导小鼠产生中和抗体特性.方法 构建2009H1N1或1918甲型流感病毒(1918H1N1)HA蛋白表达质粒2009HA和1918HA,采用25μg或200μg剂量2009HA质粒免疫小鼠,以2009HA或1918HA蛋白为包被抗原,测定小鼠血清中2009HA抗体总量或交叉反应抗体含量,分别用2009H1N1和1918H1N1两种假病毒(pp)测定抗体中和活性.结果 25 μg或200μg的2009HA质粒加强免疫小鼠后,4～16周内两组小鼠血清中2009HA总抗体水平以及对2009H1N1pp的中和抗体滴度相似(P>0.05),都含有与1918HA蛋白交叉反应抗体,对1918H1N1pp的交叉中和抗体滴度相似(P>0.05).结论 小剂量2009HA质粒DNA疫苗能够诱导小鼠产生持久的高水平中和抗体,对于预防新现流感病毒具有潜在应用价值.     

DNA聚合酶δ相互作用蛋白1能促进GFP蛋白的降解

目的证明过表达DNA聚合酶δ相互作用蛋白1(PDIP1)能否促进外源性GFP蛋白的降解。方法将HEK293细胞共转染表达质粒pEGFP-C3与pCMV-myc-PDIP1,利用荧光显微镜检测细胞绿色荧光强度,然后利用Western blot法检测GFP与PDIP1蛋白表达量的关系,再利用氯化铵与MG132分别进行干预,并检测GFP与PDIP1蛋白表达量的关系。结果共转染表达质粒pEGFP-C3与pCMV-myc-PDIP1的HEK293细胞绿色荧光强度明显低于单转pEGFP-C3质粒的细胞;随着PDIP1蛋白表达量的增大,GFP蛋白的表达量减少;利用MG132干预可以明显抑制PDIP1对GFP的降解,而氯化铵干预没有显示明显的效应。结论过表达PDIP1可以促进外源性GFP蛋白的降解,且这一降解是通过蛋白酶体途径进行的,提示PDIP1可能参与细胞内某些蛋白的泛素化降解。     

嗜肺军团菌mip基因联合疫苗诱导的小鼠免疫应答

<正>军团病主要由嗜肺军团菌引起,嗜肺军团菌共有15个血清型,其中血清1型在人军团菌病中最常见,约占80%[1],可引起严重肺炎[2]。军团菌污染的水源通过加湿器、喷雾器、淋浴器、水龙头等载体形成气溶胶。人体吸入气溶胶后引起军团菌感染[3-4]。随着人们生活水平的提高,人工冷热水系     

表达呼吸道合胞病毒G蛋白胞外区的重组H1N1流感病毒单剂滴鼻免疫可在小鼠诱导强免疫应答与保护

目的基于重组流感病毒载体的呼吸道合胞病毒(RSV)疫苗的构建及在小鼠体内的免疫保护效果评价。方法构建并拯救表达RSV A2型G蛋白胞外结构域(Gecto)的重组甲型流感病毒, 将其命名为PR8NAGecto/WSN。体外验证重组病毒G蛋白表达与病毒生长动力学后, 单剂滴鼻免疫BALB/c小鼠, 并评价体液免疫、黏膜免疫与细胞免疫。免疫4周后, 分别用RSV A2与RSV B9320进行攻毒, 通过小鼠体重变化、肺组织病毒滴度及病理评价免疫保护效果。结果单剂滴鼻免疫PR8NAGecto/WSN能在小鼠体内产生较强的体液免疫、黏膜免疫及细胞免疫。与对照组比较, 免疫组小鼠经RSV A2或B9320两个亚型病毒攻毒后肺病毒载量与肺组织病理均明显改善。结论单剂滴鼻免疫重组PR8NAGecto/WSN疫苗可在小鼠体内诱导较强的RSV特异免疫应答与攻毒保护。本研究为新型RSV黏膜疫苗的研发提供新的思路与实验资料。     

中国株HIV-1外膜蛋白基因疫苗诱导免疫小鼠免疫应答的实验研究

目的 :构建中国流行株HIV 1外膜蛋白 (gp12 0 )基因疫苗并接种小鼠 ,评价其诱导的体液和细胞免疫应答。方法 :将HIV 1gp12 0基因插入到真核表达载体pVAX1中 ,构建重组真核表达质粒pVAX1 GP12 0 ,并经EcoRI和PstI双酶切以及测序鉴定。同时以pVAX1 GP12 0和空载体pVAX1分别免疫BALB/c小鼠 ,采用ELISA检测免疫小鼠的特异性抗体和IFN γ水平 ,用MTT比色法检测免疫小鼠脾淋巴细胞的增殖 ,用乳酸脱氢酶 (LDH)试验检测小鼠特异性细胞毒性T淋巴细胞 (CTL)的应答。结果 :酶切及测序结果表明 ,成功地构建了HIV 1gp12 0基因疫苗。与空载体pVAX1组相比较 ,pVAX1 GP12 0免疫组小鼠血清抗HIV 1gp12 0抗体的滴度和IFN γ的水平均升高 ,两者差异显著 (P <0 .0 1)。pVAX1 GP12 0免疫组小鼠脾淋巴细胞增殖的刺激指数 (SI)及特异性CTL的杀伤活性 ,均高于空载体pVAX1组 (P <0 .0 1)。结论 :构建了针对我国HIV 1流行株的gp12 0基因疫苗。以其免疫BALB/c小鼠可诱导特异性体液和细胞免疫应答 ,为进一步将其用于我国的HIV的治疗奠定了基础。     

结核分枝杆菌ESAT6-CFP10融合蛋白诱导的小鼠免疫应答及其保护力

目的:研究结核分枝杆菌(MTB)ESAT6-CFP10融合蛋白诱导的小鼠体液和细胞免疫应答以及对MTB感染小鼠的保护能力。方法:用预先转移到硝酸纤维素膜条的ES-AT6-CFP10融合蛋白采用皮下包埋的方法免疫小鼠。用MTB培养上清滤液蛋白(CFP)作为抗原,用ELISA法测定免疫小鼠血清特异性抗体的滴度。末次免疫后,取部分免疫小鼠脾淋巴细胞,体外用ESAT6-CFP10融合蛋白刺激后,以MTT比色法检测淋巴细胞增殖反应,同时检测免疫小鼠IFN-γ和IL-2水平及脾细胞杀伤效应。另一部分免疫的BALB/c小鼠经尾静脉感染MTB毒株H37Rv,4周后计数脾脏中的细菌数。结果:ESAT6-CFP10融合蛋白免疫小鼠血清特异性抗体的滴度为1∶6400。淋巴细胞刺激增殖指数为1.90±0.15,明显高于生理盐水组(0.90±0.15);IFN-γ和IL-2的含量分别为1.792±19ng/L和0.211±11ng/L,显著高于生理盐水对照组,但不及BCG免疫组;同时融合蛋白诱导的脾细胞杀伤率为36%。与生理盐水免疫组(细菌负荷6.51±0.13)相比较,融和蛋白免疫的BALB/c小鼠,对攻击感染后MTB在脾脏中增殖有显著作用(细菌负荷为5.24±0.15,P<0.05),但与BCG免疫组相比脾脏中细菌负荷减少甚微。结论:ESAT6-CFP10融合蛋白可作为新型结核疫苗的候选组分。     

重组流感病毒H1N1活菌疫苗的构建及其免疫效果的初步研究

目的获得两种流感病毒重组沙门氏菌X4550(asd-pVAX1-HA)和X4550(asd-pVAX1-NA)活菌疫苗,并对其免疫效果进行初步的研究。方法将本室已构建好的重组质粒pVAX1-HA及pVAX1-NA双酶切后得到HA、NA基因,将其克隆入asd-pVAX1构建重组的表达质粒。将重组质粒转染COS-7细胞,用免疫细胞化学方法鉴定重组质粒体外的表达。进一步将重组质粒转化减毒鼠伤寒沙门氏菌X4550,以2×109CFU/只的剂量三次口服免疫Balb/c小鼠。结果asd-pVAX1-HA和asd-pVAX1-NA均能在COS-7细胞中表达;免疫小鼠不仅可以检测到HA、NA特异性的血清IgG及IgA抗体;刺激黏膜免疫反应,产生sIgA抗体,而且能抵抗40LD50同型病毒滴鼻的攻击。结论H1N1流感病毒DNA疫苗的减毒沙门氏菌运送系统在体内能够成功释放所携带的质粒,并且能够刺激机体产生保护性免疫应答。     

H3、H5、H7、H9亚型流感病毒通用多表位核酸疫苗构建及表达研究

目的构建表达A型流感病毒多种表位的核酸疫苗，并研究其在幼仓鼠肾细胞（BHK细胞）内表达产物的生物学活性。方法筛选流感病毒主要抗原（HA、NA、NP、M）表位基因，以生物信息学软件优化其排列结构，合成流感通用的复合多表位基因（Epi），以H5、H7亚型流感病毒HA融合表达基因为骨架，将多表位基因Epi插入构建H7HA-Epi-H5HA阅读盒，定向克隆至pVAX1，构建抗A型流感H3579亚型的pVAX1-H7HA-Epi-H5HA。最后将该重组质粒转染BHK细胞，提取细胞总RNA，以RT-PCR方法检测目的基因；以间接免疫荧光试验（IFA）初步测其表达产物抗原性。结果RT-PCR检测到目的基因；IFA检测到特异性荧光存在。结论本试验所构建的重组质粒pVAX1-H7HA-EpiH5HA，在真核细胞内均获了有效地转录和表达；而且其表达产物均能与相应的抗体特异性结合，证明了其具有一定的生物学活性和抗原性，有望成为抗多种A型流感病毒的通用核酸疫苗。     

Functional domains of the influenza A virus PB2 protein: identification of NP- and PB1-binding sites

Influenza virus genomic RNA segments are packaged into ribonucleoprotein (RNP) structures by the PB1, PB2, and PA subunits of an RNA polymerase and a single-strand RNA-binding nucleoprotein (NP). Assembly and function of these ribonucleoproteins depend on a complex set of protein-protein and protein-RNA interactions. Here, we identify new functional domains of PB2. We show that PB2 contains two regions that bind NP and also identify a novel PB1 binding site. The regions of PB2 responsible for binding NP and PB1 show considerable overlap, and binding of NP to the PB2 fragments could be outcompeted by PB1. The binding domains of PB2 acted as trans-dominant inhibitors of viral gene expression, and consistent with the in vitro binding data, their inhibitory activity depended on the concentration of wild-type PB2, NP, and PB1. This provides evidence for functionally significant and potentially regulatory interactions between PB2 and NP.     

A hemagglutinin-based multipeptide construct elicits enhanced protective immune response in mice against influenza A virus infection

Multipeptide constructs, comprising adjacent sequences of the 317–341 intersubunit region of immature influenza A hemagglutinin (H1N1), were designed and the functional properties of these branched peptides were compared to that of the corresponding linear peptides. In vivo studies revealed that the immunogenicity of the peptides was dependent on the presence of the hydrophobic fusion peptide (comprised in FP3), encompassing the N-terminal 1–13 sequence of the HA2 subunit. Antibody and T cell recognition, however, was directed against the 317–329 HA1 sequence, comprised in the P4 peptide. Multiple copies of P4, covalently linked by branched lysine residues, significantly enhanced the efficiency of antibody binding and the capacity of peptides to elicit B- and T-cell responses. A fraction of peptide induced antibodies reacted with immature or with proteolitically cleaved hemagglutinin (HA) molecules pretreated at low pH. Immunization with a multipeptide construct, (P4)₄–FP3, not only resulted in elevated antibody and T cell responses but conferred enhanced protection against lethal A/PR/8/34 (H1N1) infection as compared to its subunit peptides. The beneficial functional properties of this artificial peptide antigen may be acquired by multiple properties including: (i) stabilized peptide conformation which promotes strong, polyvalent binding to both antibodies and MHC class II molecules; (ii) appropriate P4 conformation for antibody recognition stabilized by the covalently coupled fusion peptide, resulting in the production of virus cross reactive antibodies which inhibit the fusion activity of the virus; (iii) activation of peptide specific B cells which potentiate antigen presentation and peptide specific T cell responses; and (iv) generation of helper T cells which secrete lymphokines active in the resolution of infection.     

Immunization with a low dose of hemagglutinin-encoding plasmid protects against 2009 H1N1 pandemic influenza virus in mice

A vaccine against the novel pandemic influenza virus (2009 H1N1) is available, but several problems in preparation of vaccines against the new emerging influenza viruses need to be overcome. DNA vaccines represent a novel and powerful alternative to conventional vaccine approaches. To evaluate the ability of a DNA vaccine encoding the hemagglutinin (HA) of 2009 H1N1 to generate humoral responses and protective immunity, BALB/c mice were immunized with various doses of 2009 H1N1 HA-encoding plasmid and anti-HA total IgG, hemagglutination inhibition antibodies and neutralizing antibodies were assayed. The total IgG titers against HA correlated positively with the doses of DNA vaccine, but immunization with either a low dose (10 μg) or a higher dose (25-200 μg) of HA plasmid resulted in similar titers of hemagglutination inhibition and neutralizing antibodies, following a single booster. Further, 10 μg plasmid conferred effective protection against lethal virus challenge. These results suggested that the DNA vaccine encoding the HA of 2009 H1N1 virus is highly effective for inducing neutralizing antibodies and protective immunity. DNA vaccines are a promising new strategy for the rapid development of efficient vaccines to control new emerging pandemic influenza viruses.     

Protective immunity against H5N1 influenza virus by a single dose vaccination with virus-like particles

We generated influenza virus-like particles (VLPs) containing the wild type (WT) H5 hemagglutinin (HA) from A/Viet Nam/1203/04 virus or a mutant H5 HA with a deletion of the multibasic cleavage motif. VLPs containing mutant H5 HA were found to be as immunogenic as VLPs containing WT HA. A single intramuscular vaccination with either type of H5 VLPs provided complete protection against lethal challenge. In contrast, the recombinant H5 HA vaccine was less immunogenic and vaccination even with a 5 fold higher dose did not induce protective immunity. VLP vaccines were superior to the recombinant HA in inducing T helper type 1 immune responses, hemagglutination inhibition titers, and antibody secreting cells, which significantly contribute to inducing protective immunity after a single dose vaccination. This study provides insights into the potential mechanisms of improved immunogenicity by H5 VLP vaccines as an approach to improve the protective efficacy against potential pandemic viruses.     

抗三价流感病毒亚单位疫苗IgY防治小鼠流感病毒感染的实验研究

目的:探讨抗三价流感病毒亚单位疫苗IgY(Immunoglobulin Yolk)预防及治疗流感病毒感染小鼠的作用。方法:建立A/fm/1/47(H1N1)流感小鼠模型;分5组,观察各组小鼠死亡率,脾、肺指数及肺指数抑制率。结果:0.2%抗三价流感病毒亚单位疫苗IgY于攻毒前24、6、1小时滴鼻3次显著地预防流感病毒致死率(30.0%),而攻毒后1、6、24小时分别滴鼻3次,此后每天滴鼻1次,持续5天,致死率为20.0%,两组与阳性对照组(致死率90.0%)、非特异性IgY预防和治疗组(致死率80.0%,70.0%)比较差异显著(P<0.01)。0.2%抗三价流感病毒亚单位疫苗IgY预防组和治疗组肺指数显著低于阳性对照组(P<0.05~0.01),其治疗组也低于非特异性IgY预防和治疗组(P<0.05)。结论:抗三价流感病毒亚单位疫苗IgY感染前后24小时内多次滴鼻能有效地预防和治疗流感病毒感染。