猪细小病毒VP2与大肠杆菌不耐热肠毒素B亚单位在干酪乳杆菌表面共表达

将分别编码猪细小病毒(PPV)主要免疫保护性抗原VP2蛋白与大肠杆菌不耐热肠毒素B亚单位(LTB)基因插入乳酸杆菌细胞表面表达载体pPG中, 成功构建了重组表达载体pPG-VP2-LTB, 将其电转化干酪乳杆菌Lactobacillus casei 393, 获得了表达猪细小病毒VP2-LTB融合蛋白的重组乳酸菌表达系统, 经2%乳糖诱导, SDS-PAGE和Western-blot检测表明, 有大小约78 kD的蛋白得到了表达, 具有与天然病毒蛋白一样的抗原特异性, 全细胞ELISA结果表明, LTB同     

猪瘟病毒T细胞表位E290多肽与猪细小病毒VP2蛋白在干酪乳杆菌中的共表达及免疫小鼠特异性抗体的测定

将分别编码猪瘟病毒T细胞表位E290多肽和猪细小病毒主要保护性抗原VP2蛋白的重组基因插入干酪乳杆菌分泌型表达载体pPG中,构建了重组表达载体pPG-VP2-E290,将其电转化干酪乳杆菌Lactobacillus casei 393,获得了猪瘟病毒T细胞表位E290多肽与猪细小病毒VP2蛋白的乳酸菌共表达系统,经2%乳糖在MRS培养基中的诱导表达,对诱导表达的菌体及培养上清液进行SDS-PAGE检测表明,有约70kDa蛋白得到了表达,表达蛋白的大小与理论值相符。Western blot分析结果表明所表达的蛋白具有与天然病毒蛋白一样的抗原特异性。以诱导表达上清液作为抗原进行的间接ELISA实验也表明,重组的目的蛋白获得了分泌表达。将该重组干酪乳杆菌经口服接种途径免疫BALB/c小鼠,收集粪便样品检测小鼠产生抗PPV的特异性sIgA抗体,采集血液样本检测血清中抗PPV及抗E290的特异性IgG。结果表明分泌型的重组菌pPG-VP2-E290/L.casei 393免疫小鼠能够产生明显的抗体水平,为重组猪瘟与猪细小病毒乳酸菌口服活菌疫苗的研制奠定了重要的物质基础。     

猪乳铁蛋白在4种重组乳杆菌中的表达及抑菌活性比较

为了获得优化的猪乳铁蛋白乳杆菌表达系统,并比较重组猪乳铁蛋白的抑菌活性,根据乳杆菌使用密码子的偏嗜性优化合成猪乳铁蛋白成熟肽编码序列,将其克隆到乳杆菌表达载体pPG612.1的XhoⅠ/BamHⅠ位点,获得了plf乳杆菌表达载体质粒pPG612.1-plf。将获得的重组质粒分别电转化入干酪乳杆菌ATCC393、戊糖乳杆菌KLDS1.0413、植物乳杆菌KLDS1.0344和副干酪乳杆菌KLDS1.0652细胞内,获得4种表达猪乳铁蛋白的重组乳杆菌。经木糖诱导,通过Western blotting和激光共聚焦检测重组猪乳铁蛋白的表达,用ELISA方法检测和比较4种重组菌上清中表达猪乳铁蛋白的量,并用琼脂孔穴扩散抑菌法检测4种重组乳杆菌表达乳铁蛋白的抑菌活性。结果表明,乳铁蛋白在4种重组乳杆菌中均得到正确表达,其产物分子量约73 kDa,重组干酪乳杆菌、重组戊糖乳杆菌、重组植物乳杆菌和重组副干酪乳杆菌的重组猪乳铁蛋白表达量分别为9.6μg/mL、10.8μg/mL、12.5μg/mL、9.9μg/mL。重组猪乳铁蛋白对大肠杆菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、巴氏杆菌和李氏杆菌均有一定的抑菌作用,对金黄色葡萄球菌的抑菌作用最强,且4种重组乳杆菌中重组植物乳杆菌表达产物的抑菌效果优于其他重组菌的表达产物。结果表明在4种乳杆菌中重组猪乳铁蛋白的最佳表达系统为植物乳杆菌,该结果为猪乳铁蛋白的乳杆菌表达系统进一步开发与应用奠定了基础。     

重组ETEC k88ac-LTB干酪乳杆菌在人工消化液中的生存性能

探讨重组干酪乳杆菌(Lactobacillus casei)在人工消化液中的存活能力。将K88ac-LTB基因从表达载体pQE-30克隆到L. casei细胞表面表达载体pLA中, 构建了重组表达载体pLA-K88ac-LTB, 并将其电转化至L. casei中, 在MRS培养基中培养后, Western blotting检测, 有约71.2 kD蛋白得到了表达, 表达蛋白的大小与理论值相符且可被抗血清所识别, 间接免疫荧光实验及流式细胞结果表明, 多聚谷氨酸合成酶A蛋白(pgsA)能够将融合蛋白成功地展示在菌体表面。将重组菌接种于人工模拟的胃肠液中, 通过平板计数观察其存活能力。结果表明, 重组干酪乳杆菌在人工消化液中均具有良好的存活性能, 符合益生菌的基本特征, 为实验动物模型的建立奠定了基础。     

犬细小病毒VP2蛋白在真核细胞中的分泌表达及特性

摘要:【目的】利用真核细胞分泌表达犬细小病毒VP2蛋白和研究其特性。【方法】为构建犬细小病毒（Canine parvovirus, CPV）VP2基因的真核分泌型表达载体,首先通过酶切从含有人CD5信号肽序列的质粒中将CD5信号肽基因片段切出,将其连接到真核表达载体pcDNA3.1A的多克隆位点上,构建成pcDNA3.1-CD5sp质粒。然后再通过PCR方法从含有犬细小病毒VP2基因的质粒中扩增VP2基因,并将其插入到pcDNA3.1- CD5sp载体中CD5信号肽的下游,构建成VP2基因的真核分泌型表达载体pcDNA-CD5sp-VP2。经磷酸钙介导转染293T细胞,使其在真核细胞中进行分泌表达,并通过ELISA检测表达的VP2蛋白与犬转铁蛋白受体（TfR）结合的活性。【结果】序列分析结果表明,本实验构建的犬细小病毒VP2基因真核分泌型表达载体结构正确,将该表达载体转染的293T细胞,在培养基中通过Western-blot检测到有VP2重组蛋白的存在。经ELISA检测表明表达的重组VP2蛋白具有与犬转铁蛋白受体结合的活性。【结论】 利用人的CD5信号肽实现了犬细小病毒VP2蛋白在真核细胞中的分泌表达,表达的VP2蛋白具有与犬转铁蛋白受体结合的活性。     

犬细小病毒VP2基因的克隆及其在毕赤酵母中的表达

目的表达犬细小病毒VP2蛋白（CPVVP2）,用于犬细小病毒病的诊断、疫苗研制和VP2蛋白功能研究。方法采用PCR方法对CPVVP2基因进行扩增,将CPVVP2基因克隆到毕赤酵母（Pichiapastoris）分泌表达载体pPICZαA中,构建真核重组表达载体pPICZαA—VP2,将该重组质粒线性化后,转化毕赤酵母菌GS115中,在甲醇诱导下表达CPVVP2,SDS-PAGE和Western blotting鉴定表达蛋白。结果成功扩增了CPVVP2基因,构建了真核重组表达载体pPICZαA-VP2在毕赤酵母菌中表达出约64．35kD蛋白。Western blotting鉴定表明,表达VP2蛋白与犬细小病毒阳性血清有反应性。结论在毕赤酵母中成功地表达了CPVVP2蛋白,能被犬细小病毒阳性血清识别。     

表面表达猪流行性腹泻病毒核蛋白蛋白的重组干酪乳杆菌诱导产生的免疫应答

为探讨表达猪流行性腹泻病毒(PEDV)核蛋白(N)基因的重组干酪乳杆菌口服免疫小鼠后诱导特异性免疫应答,本研究制备表达流行性腹泻病毒核蛋白的重组干酪乳杆菌,应用Western blotting、间接免疫荧光和全细胞ELISA鉴定目的蛋白的表达。然后用该重组干酪乳杆菌口服免疫BALB/c小鼠,分别测定了免疫后不同时间血清中特异性IgG、粪便中特异性的sIgA水平以及血清的中和活性;并测定免疫小鼠脾淋巴细胞增殖情况和细胞因子水平。结果显示,目的蛋白表达在细胞表面,可被阳性血清所识别。免疫小鼠后,可分别在血清中和粪便中检测到较高水平特异性IgG、sIgA(P<0.01),但血清并没有中和活性;淋巴细胞增殖试验和细胞因子测定结果显示,免疫组可产生明显的细胞免疫应答。结果表明,该重组干酪乳杆菌表达系统可诱导小鼠产生黏膜免疫应答和系统免疫应答,具有作为口服疫苗潜在的应用价值。     

猪细小病毒核酸疫苗的构建及其对小鼠免疫原性的研究

将猪细小病毒VP2基因克隆至pCI-neo真核表达载体中,构建了pCIneo-VP2重组质粒,转染至PK-15细胞中,利用免疫荧光方法检测在体外表达情况;并以小鼠为动物模型,将pCIneo-VP2、pCI-neo重组质粒、猪细小病毒活疫苗和对照组通过肌肉注射进行免疫,检测免疫小鼠的淋巴细胞转化功能,特异性CTL杀伤活性和血清抗体滴度。结果显示,pCIneo-VP2在体外能够诱导PK-15细胞表达VP2蛋白,小鼠注射pCIneo-VP2质粒 1周后能够诱导机体产生抗体,4周时达到峰值,与活疫苗对照组产生的抗体滴度、诱导T淋巴细胞增殖和诱导强的细胞毒性基本一致。试验表明,构建的pCIneo-VP2能够有效诱导机体产生体液免疫和细胞免疫,为研制出高效、新型猪细小病毒疫苗提供了科学依据和试验依据。     

表达猪细小病毒VP2基因的重组伪狂犬病毒的构建及其生物学特征研究

根据Bergeron等报道的猪细小病毒(PPV)基因组,设计一对包含VP2全基因的PCR引物,上下游均引入一个BamHI位点,扩增得到VP2基因后,将其插入到pUSK载体中,构建了转移载体pUSK-VP2.采用脂质体介导的转染方法,将伪狂犬病毒TK-/gG-/LacZ 株的基因组DNA与pUSK-VP2共转染PK-15细胞,待细胞病变后收集病毒液,在空斑纯化的同时,利用检测PPV VP2基因和LacZ基因的PCR方法筛选重组病毒TK-/gG-/VP 2株,Southern blotting、SDS-PAGE、Western blotting和电镜观察鉴定重组病毒,并在不同细胞上测定重组病毒的增殖滴度,接种小鼠进行安全性试验.结果发现,外源基因VP2已成功地插入到TK-/gG-/LacZ 亲本株的基因组中,并获得了表达.表达的VP2蛋白可以与猪细小病毒阳性血清反应,而且可以自行装配成病毒样颗粒.同时发现,VP2基因的插入不影响重组病毒的增殖特性,其毒力与亲本株相当.     

短小芽孢杆菌β-1，4-甘露聚糖酶的酶学性质及其在干酪乳杆菌中的表达

摘要:【目的】干酪乳杆菌广泛的应用于食品加工和饲料行业,本研究拟构建表达甘露聚糖酶的重组干酪乳杆菌并进行相关评价。【方法】利用干酪乳杆菌表达载体pELX1和pELSH,将短小芽孢杆菌的β-1,4-甘露聚糖酶成熟肽的基因克隆到上述两个载体中,构建的重组质粒电转化到干酪乳杆菌宿主中,分别构建能够胞内表达和分泌表达甘露聚糖酶的重组干酪乳杆菌。【结果】重组干酪乳杆菌菌株经培养后,胞内表达的β-1,4-甘露聚糖酶在重组细胞总蛋白中最高可达23 U/mg,分泌表达培养基上清的β-1,4-甘露聚糖酶最高达到8.8 U/mL。【结论】本研究首次实现了甘露聚糖酶在干酪乳杆菌中的表达,结果表明该重组干酪乳杆菌具有较大的应用前景,值得进一步研究。     

表达重组猪瘟病毒E290多肽的干酪乳杆菌口服免疫特性及诱导特异性CTL反应的研究

经PCR扩增获得约60bp编码猪瘟病毒T细胞表位E290多肽基因片段,克隆至表达载体pPG-VP2中VP2基因5′端上游,命名为pPG-VP2-E290,电转化干酪乳杆菌,构建了表达猪瘟病毒E290多肽的重组乳酸菌系统。口服免疫BALB/c鼠和新西兰兔,检测诱导小鼠和兔体内产生特异性抗猪瘟病毒E290多肽IgG水平,并对E290多肽的CTL活性进行检测,同时对免疫兔进行猪瘟病毒攻毒实验,检测E290多肽抗体对免疫兔的保护作用。构建的重组猪瘟病毒T细胞表位的干酪乳杆菌具有良好的免疫性,口服免疫后的小鼠和兔血清中均检测到了较高水平的抗E290多肽抗体IgG,且能诱导小鼠机体产生抗猪瘟病毒的特异性CTL反应,亦证实猪瘟病毒E290免疫兔能够抵抗猪瘟病毒的攻击。     

Induction of immune responses in mice after intragastric administration of Lactobacillus casei producing porcine parvovirus VP2 protein

Lactobacillus casei ATCC 393 was selected as an antigen delivery vehicle for mucosal immunization against porcine parvovirus (PPV) infection. A 64-kDa fragment of PPV major protective antigen VP2 protein was used as the parvovirus antigen model. A recombinant Lactobacillus expressing VP2 protein was constructed with plasmid pPG611.1, where expression and localization of the VP2 protein from recombinant Lc393-rPPV-VP2 was detected via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and immunofluorescence. Both local mucosal and systemic immune responses against PPV were induced in BALB/c mice immunized orally with the recombinant Lactobacillus expressing VP2 protein. The induced antibodies demonstrated neutralizing effects on PPV infection. These data indicated that the use of recombinant lactobacilli could be a valuable strategy for future vaccine development of PPV.     

Induction of Immune Responses in Mice after Intragastric Administration of Lactobacillus casei Producing Porcine Parvovirus VP2 Protein

Lactobacillus casei ATCC 393 was selected as an antigen delivery vehicle for mucosal immunization against porcine parvovirus (PPV) infection. A 64-kDa fragment of PPV major protective antigen VP2 protein was used as the parvovirus antigen model. A recombinant Lactobacillus expressing VP2 protein was constructed with plasmid pPG611.1, where expression and localization of the VP2 protein from recombinant Lc393-rPPV-VP2 was detected via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and immunofluorescence. Both local mucosal and systemic immune responses against PPV were induced in BALB/c mice immunized orally with the recombinant Lactobacillus expressing VP2 protein. The induced antibodies demonstrated neutralizing effects on PPV infection. These data indicated that the use of recombinant lactobacilli could be a valuable strategy for future vaccine development of PPV.     

Production of porcine parvovirus virus-like particles using silkworm larvae

Porcine parvovirus (PPV) is a significant causative agent of porcine reproductive failure, causing serious economic losses in the swine industry. PPV is a nonenveloped virus, and its capsid is assembled from three viral proteins (VP1, VP2, and VP3). The major capsid protein, VP2, is the main target for PPV neutralizing antibodies and vaccine development. In this study, PPV-VP2 protein was expressed in silkworm larvae, and its antigenicity and production were compared with those in B. mori cells (Bm5). The recombinant VP2 protein was expressed successfully in silkworm larvae and Bm5 cells with a size of approximately 64 kDa. The formation of virus-like particles (VLPs) by recombinant PPV-VP2 was confirmed through transmission electron microscopy. The recombinant PPV-VP2 protein assembled into spherical particles with diameters ranging from 20 to 22 nm. The antigenicity of PPV-VLPs was comparatively analyzed between Bm5 cells and silkworm larvae by ELISA, hemagglutination and hemagglutination inhibition assays. Consequently, it was confirmed that the PPV-VLPs produced in the silkworm larvae were more antigenic than VLPs produced in Bm5 cells. Therefore, it is expected that economical and effective vaccine development will be possible by mass production of PPV-VLPs in silkworm larvae.     

A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

Background

Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene.

Methods

A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein.

Results

Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts.

Conclusions

In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

     

表面展示新城疫病毒部分F蛋白的重组干酪乳杆菌的构建及其免疫原性

本研究旨在构建重组干酪乳杆菌pLA-Newcastlediseasevirus (NDV)-F/Lactobacillus casei,获得表达产物,并探讨其免疫效果。利用PCR扩增携带部分主要抗原表位的NDV F基因,与穿梭质粒pLA连接转化至大肠杆菌BL21 (DE3)中,筛选阳性重组质粒,将其电转化至干酪乳杆菌中,构建重组干酪乳杆菌pLA-NDV-F/L. casei,应用PCR鉴定阳性菌株,Western blotting鉴定重组菌反应原性,间接免疫荧光、流式细胞术和激光共聚焦检测蛋白表达情况。试验选用14日龄雏鸡,各组免疫方式为口服+滴鼻。设立pLA-NDV-F/L. casei两次免疫组和三次免疫组、弱毒疫苗组、 pLA/L.casei、未攻毒PBS组和攻毒PBS组。间接ELISA方法检测雏鸡血清IgG、肠道、鼻腔、肺脏中sIgA抗体效价,评价试验组雏鸡攻毒保护率。结果表明,有94.10%的重组菌表达了F蛋白,且高效表达在干酪乳杆菌细胞表面,蛋白大小为62kDa,并能与抗NDV阳性血清特异性结合。各免疫组anti-F IgG和s Ig A抗体水平显著高于对照组,p LA-NDV-F/L. casei三次免疫组抗体持续时间比两次免疫组延长28 d,抗体峰值没有显著差异。免疫pLA-NDV-F/L. casei三次、两次、弱毒疫苗、pLA/L. casei和PBS的攻击保护率分别为80%、80%、90%、0%和0%。因此,利用干酪乳杆菌表达体系成功表达了携带部分抗原表位的NDVF基因,具备良好的反应原性和免疫原性,可诱导机体产生保护性免疫应答。     

Lactobacillus casei磷脂酶A_2基因在大肠杆菌中的重组表达

以Lactobacillus casei染色体基因组为模板,PCR扩增获得磷脂酶A2基因pla2,以pET-28a(+)为载体构建重组表达质粒pET-28a(+)-pla2。通过IPTG诱导实现磷脂酶A2在E.coli DE3中的重组表达。对诱导条件初步优化后,重组菌酶活最大可达2.8 U/mL。通过Ni-螯合柱对目的蛋白进行纯化,SDS-PAGE分析重组磷脂酶A2相对分子质量为1.7×104。通过酶学性质分析,最适温度为37℃,最适pH 8,比酶活为110 U/mg。