利用S-层蛋白在细胞表面展示α-淀粉酶和金属硫蛋白

选用苏云金芽胞杆菌CTC菌株细胞表面S 层蛋白作为表面展示载体 ,利用其N 端信号肽以及锚定序列分别将不同生物特性的外源蛋白带到胞外并锚定在细胞的表面。为研究外源蛋白的分子量和生化特性对表面展示以及对其在细胞表面生物活性的影响 ,选用分子量较大的分泌性蛋白α 淀粉酶和富含半胱氨酸的非分泌性蛋白金属硫蛋白作为目标蛋白。将α 淀粉酶的结构基因 (amy)和金属硫蛋白的结构基因 (smtA)与S 层蛋白的锚定区slh结合 ,构建融合蛋白基因slh amy及slh smtA ,克隆至穿梭载体pHT30 4上 ,得到重组质粒pBMBSA 30 4和pBMBSM 30 4 ;转入带有帮助质粒的重组菌株BMB171 pBMB CSA中 ,得到新的重组菌株BMBSAC和BMBSMC。SDS PAGE显示融合蛋白SLH AMY和SLH SMTA在重组菌的表面得到了表达。利用锥虫蓝染色 打孔加膜法表明重组菌SAC的确具有表面酶活性 ,α 淀粉酶被固定在细胞表面。碘液法测定的数据表明重组菌SAC的菌悬液样品较之受体菌BMB171菌悬液样品酶活提高了 4 2 4 %。Ni NTA 琼脂糖微球吸附试验显示重组菌SMC能够被微球所吸附 ,证实SMTA在重组菌表面具有活性。对游离镉离子的吸附试验显示重组菌对镉离子的吸附能力是对照受体菌的 4倍。     

禽流感病毒核蛋白在噬菌体表面的展示

利用PCR技术,扩增禽流感病毒（AIV）核蛋白基因片段,将其克隆至pR质粒的T4噬菌体SOC基因C末端获得重组质粒pR—np,以此重组载体转化大肠杆菌Escherichia coli2（E2）,用溶菌酶缺陷噬菌体T4-zl感染重组E2菌,重组载体与缺陷噬菌体T4-zl基因发生同源重组,将np基因整合到噬菌体基因组中,用PCR方法筛选重组噬菌体并命名为T4-zl—np。经Western blot免疫电镜与ELISA检测证实,T4-zl-np表达的NP融合蛋白具有免疫学活性。结果表明,AIV核蛋白成功地在T4噬菌体表面展示,为禽流感防治奠定了基础。     

苏云金芽胞杆菌LM1212菌株cry基因启动子的活性分析

【背景】前期发现一株具有独特分化表型的苏云金芽胞杆菌(Bacillusthuringiensis,Bt)LM1212,该菌株共有14种杀虫基因,组成了10个转录单元。利用源自菌株LM1212的晶体产生细胞调控因子(crystal producing cell regulator, CpcR)以及其可以激活的cry35-like基因启动子,已在典型Bt菌株HD73中成功构建了非芽胞杀虫蛋白表达体系。【目的】比较菌株LM1212的不同杀虫基因启动子的转录活性,明确可被转录因子CpcR激活的且转录活性较高的启动子,以此为基础优化非芽胞杀虫蛋白表达体系。【方法】将10个启动子区域分别与lacZ报告基因融合构建在pHT304-18Z载体上,得到了10个重组质粒;然后将cpcR基因及其启动子(PcpcR-cpcR)分别反向构建在选取的启动子区域与lacZ报告基因的上游,得到可以表达CpcR且与上述构建相对应的10个重组质粒,将这些重组质粒分别转入不含CpcR的菌株HD73,即获得20个可用于测定β-半乳糖苷酶活性的重组菌株。通过光学显微镜观察和SDS-PAGE检测明确杀虫蛋白表达的情况。【结果】在...     

杆状病毒表面展示甲型流感病毒核蛋白的免疫原性及保护效果的初步评价

流感病毒的核蛋白(Nucleoprotein,NP)高度保守,具有型特异性,能够诱导产生细胞介导的免疫应答,从而起到保护作用,抵抗不同亚型流感病毒的攻击。本研究利用杆状病毒表面展示技术,将杆状病毒GP64蛋白的信号肽(Signal peptide,SP)和胞质尾(Cytoplasmic tail,CT)与甲型流感病毒(A/Hubei/1/2010(H5H1))的NP蛋白融合表达,从而将NP蛋白展示在杆状病毒的表面,并对该病毒NP蛋白的免疫原性和保护效果进行了研究。Westernblot实验证明NP蛋白可以在Sf9细胞中有效表达。免疫电镜试验显示NP蛋白已成功展示在杆状病毒表面。通过小鼠感染实验,ELISA结果证明NP疫苗可以诱导产生高水平的IgG血清抗体。ELISPOT结果表明NP蛋白的2个细胞免疫抗原表位(NP57-66IQNSITIERM和NP441-450RTEIIKMMES)可以诱导小鼠脾淋巴细胞产生IFN-γ,利用NP57-66、NP441-450和NP蛋白均可刺激CD4+和CD8+T细胞产生IFN-γ,但主要以CD8+诱导为主,表明重组病毒可以产生有效的细胞免疫反应。利用小鼠鼠肺适应株A/PR/8/34(H1N1)进行攻毒实验,结果表明该重组病毒感染小鼠后可以降低小鼠肺病毒载量,减轻肺组织损伤,减少体重下降,并可以保护50%小鼠存活。     

罗非鱼无乳链球菌Sip基因的克隆、表达及免疫原性分析

表面免疫原性蛋白(Surface Immunogenic Protein,Sip)是B群链球菌(Group B Streptococcus,GBS)的一种表面蛋白,在GBS多种血清型的菌株中均有表达。研究从实验室分离到的罗非鱼无乳链球菌广东株的基因组DNA中扩增出Sip基因,构建原核表达载体pColdII-Sip,转化大肠杆菌(Escherichia coli)BL21(DE3)菌株。经诱导表达、SDS-PAGE电泳检测显示重组蛋白主要以可溶形式表达。重组蛋白用亲和层析的方法纯化,蛋白纯度达98%。纯化后的重组蛋白免疫罗非鱼(Oreochromis niloticus,GIFT strain)以分析其免疫原性。受免鱼免疫14d后进行人工攻毒试验,免疫组的相对保护率为70%—87%。酶联免疫吸附试验(Elisa)显示受免鱼对重组蛋白产生了较好的免疫应答,免疫剂量为3μg/g和5μg/g时受免鱼血清抗体滴度可达1:128000。研究结果显示重组蛋白Sip具有较强的免疫原性和保护作用,Sip基因可作为罗非鱼链球菌基因工程亚单位疫苗候选基因。     

表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌对小鼠的免疫原性

【目的】构建表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌,对重组菌株的生物学特性以及对小鼠的免疫原性进行研究。【方法】分别将猪肺炎支原体的免疫原性基因p36、p46、p65和p97R1-Nrdf克隆到pYA3493,得到重组质粒pYA-36、pYA-46、pYA-65和pYA-97R1-Nrdf。重组质粒和空质粒pYA3493分别电转asd基因缺失株C500ˉ,获得重组菌株C36(pYA-36)、C46(pYA-46)、C65(pYA-65)、C97R1-Nrdf(pYA-97R1-Nrdf)和空质粒菌株CpYA(pYA3493)。研究重组菌株的生物学特性,并以小鼠为动物模型评价重组菌株在口服、肌注两种不同免疫途径下的免疫原性。【结果】成功构建表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌,重组菌株能表达外源蛋白,生化和生长特性未发生改变,插入的外源基因亦稳定存在。小鼠的免疫原性结果显示:口服C36+C46+C65+C97R1-Nrdf组的猪肺炎支原体抗体极显著高于口服C36+C46+C65组和肌注商品疫苗组(P<0.01),但与肌注C36+C46+C65组无显著性差异(P>0.05);IFN-γ为肌注C36+C46+C65组显著高于肌注商品疫苗组(P<0.05),而与口服C36+C46+C65或C36+C46+C65+C97R1-Nrdf组差异均不显著(P>0.05);IL-4水平为口服C36+C46+C65组>口服C36+C46+C65+C97R1-Nrdf组>肌注商品疫苗组>肌注C36+C46+C65组,但各组之间差异均不显著(P>0.05)。对照组的猪肺炎支原体抗体、IFN-γ以及IL-4均与试验组差异极显著(P<0.01)。【结论】构建的表达猪肺炎支原体免疫原性基因的重组猪霍乱沙门氏菌,采用肌注免疫时具有较好的免疫原性,有望发展为猪肺炎支原体的基因工程疫苗。     

苏云金杆菌辅助蛋白基因p19和orf1-orf2对杀虫晶体蛋白CytlAa表达的影响

为检测苏云金杆菌辅助蛋白P19和0RFl—0RF2对杀虫晶体蛋白CytlAa表达的影响,构建了5个重组表达质粒。5个质粒都含有cytlAa基因,但pT1只含有cytlAa基因,p他同时含有p19基因,pt3同时含有orf1-orf2串联基因,pT4同时含有p19基因和p20基因,pT5同时含有orf1-orf2串联基因和p20基因。将这5个表达质粒和质粒pWF45电转化到苏云金杆菌晶体缺陷型4Q7中,分别获得转化菌株Bt—T1、Bt—T2、Bt—T3、Bt—T4、Bt—T5和Bt—WF45。SDS—PAGE结果显示,菌株Bt—T1、Bt—T2和Bt—T3只产生少量的27kD cytlAa蛋白,而且部分降解为大约24kD的蛋白。而Bt—T4和Bt—T5能产生大量的cytlAa蛋白,但Bt—T4和Bt—T5的cytlAa蛋白产量都明显少于Bt—WF45。电镜观察和生物测定结果表明Bt—T4和Bt—T5与Bt—WF45的晶体大小和杀蚊毒力没有显性差异。研究表明p19和ORF1—ORF2对CytlAa。蛋白的合成显示可能有抑制作用。     

轮状病毒VP4抗原表位在VP6载体蛋白同一位点表达比较研究

目的:评价轮状病毒(RV)VP4两个抗原表位插入VP6载体蛋白同一位点所表达的重组嵌合蛋白免疫学性质及在研制嵌合蛋白疫苗中的意义。方法:采用分子克隆和基因重组技术将RV VP4的两个抗原表位插入到VP6载体蛋白同一位点上,构建重组抗原表达质粒,表达携带不同抗原表位的重组嵌合蛋白,用Western blot和中和试验分析重组嵌合蛋白的抗原反应性和免疫原性。结果:成功构建了两个嵌合蛋白表达质粒,并在大肠杆菌中高效表达;表达的嵌合蛋白可与相应抗体特异性反应;可诱导豚鼠产生特异性血清抗体;抗嵌合蛋白血清抗体可特异性识别载体蛋白VP6F,Wa株病毒的VP6和VP4蛋白,可中和Wa株病毒在MA104细胞上的感染性;结果表明,所构建和表达的两个以VP6为载体的VP4抗原表位嵌合蛋白具有较高抗原反应性和免疫原性;嵌合蛋白携带的VP4抗原表位具有增强载体蛋白免疫原性作用;为研制新型RV重组蛋白疫苗的奠定了较好的基础。     

登革3型病毒prM-E基因重组质粒DNA的构建和真核表达

构建登革 3型病毒 prM E基因的真核表达重组质粒 ,并进行体外表达 ,为登革DNA疫苗的研究奠定基础。用RT -PCR法获得 prM -E基因片段 ,然后将其克隆到真核表达载体中。用电穿孔法将重组质粒DNA转入BHK细胞 ,通过免疫荧光法检测外源基因在真核细胞中的表达。结果 ,通过酶切和序列测定证实了构建的重组质粒DNA含序列正确的 prM- E基因。用免疫荧光法检测到转染了重组质粒DNA的BHK细胞的胞浆中有登革 3型病毒特异蛋白的表达。说明含有登革 3型病毒prM -E基因的真核表达重组质粒可以在BHK细胞中表达 ,该结果为观察该重组质粒的免疫原性奠定了基础。     

一种展示SARS冠状病毒受体结合区的重组枯草杆菌芽孢的制备及免疫原性分析

目的：利用枯草杆菌芽孢呈递技术制备表达SARS冠状病毒S蛋白受体结合区（RBD）的重组芽孢。方法：将枯草杆菌 CotB 基因构建到基因组整合质粒pDG1664中,再将 RBD 基因连接到 CotB 基因的下游,构建成重组质粒pDG1664-CotB-RBD,通过同源重组整合到PY-79枯草杆菌基因组中;利用红霉素抗性筛选重组菌并进行PCR和DNA测序鉴定,Western印迹鉴定重组菌芽孢表面RBD蛋白的表达情况;用表达RBD的重组芽孢以口服方式免疫小鼠,通过ELISA和流式细胞术检测重组芽孢的免疫原性。结果：制备出枯草杆菌基因组整合了RBD抗原基因的重组菌株RS1931,形成的重组芽孢表达相对分子质量约62×103的CotB-RBD融合蛋白;重组芽孢免疫的小鼠血清RBD抗原特异性IgG抗体滴度在末次免疫后2周可达1∶10880,重组芽孢初免后18周的小鼠脾细胞中IFN-γ＋CD4^＋、IL-4＋CD4^＋和IFN-γ＋CD8^＋T细胞比例上调,表明重组芽孢经口服免疫产生良好的体液免疫和细胞免疫应答。结论：针对SARS冠状病毒S蛋白RBD建立了枯草杆菌芽孢呈递技术方法,制备出在枯草杆菌芽孢表面稳定表达外源RBD蛋白的重组株,获得的重组芽孢具有良好的免疫原性,为开发芽孢呈递型SARS疫苗奠定了基础。     

Display of avian influenza virus nucleoprotein on <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> cell surface using CTC as a fusion partner

The S-layer protein CTC surface display system of Bacillus thuringiensis was used to test the possibility of displaying avian influenza virus nucleoprotein (NP) on the cell surface of B. thuringiensis. By fusing np with the anchoring motif of ctc, four recombinant plasmids were constructed. They harbored fusion gene ctc-np, csa-ctc-np (csa representing csaAB operon, very important in anchoring S-layer protein on cell surface), ctc-npp (npp representing the part fragment of np), and csa-ctc-npp, respectively. Five recombinant strains were obtained by transferring recombinant plasmids to B. thuringiensis plasmid-free derivative strain BMB171. The vegetative cells of five strains were used as agglutinogens for slide agglutination assays. The assays showed recombinant NP proteins successfully displayed on the cell surface of five strains. After immunization of chickens with spores by oral route, all five strains elicited a humoral response to NP and exhibited immunogenicity as indicated by enzyme-linked immunosorbent assay (ELISA). ELISA also showed that one of five strains, CN (bearing csa-ctc-npp), exhibited the highest immunogenicity among five strains, which suggested that the best way of constructing ctc fusion gene was the csa-ctc-npp. The strategy developed in this study suggests the possibility of generating a heat-stable and oral veterinary vaccine with B. thuringiensis surface display system.     

利用S-层蛋白在苏云金芽胞杆菌细胞表面展示多聚组氨酸肽（简报）

S层(Slayer)是由单一的蛋白或糖蛋白组成的薄层晶状结构,它广泛存在于古细菌和真细菌细胞的最外表面,可包裹整个细胞。S层在结构化学、形态学、遗传学以及物理化学等方面具有独特的性质,使之在生物技术、分子纳米技术和仿生学等领域蕴藏着广泛的应用潜力。近年来,随着微生物表     

利用S-层蛋白在苏云金芽胞杆菌细胞表面展示多聚组氨酸肽

S-层(S-layer)是由单一的蛋白或糖蛋白组成的薄层晶状结构,它广泛存在于古细菌和真细菌细胞的最外表面,可包裹整个细胞。S-层在结构化学、形态学、遗传学以及物理化学等方面具有独特的性质,使之在生物技术、分子纳米技术和仿生学等领域蕴藏着广泛的应用潜力。近年来,随着微生物表面展示技术的兴起和展示系统的逐渐成熟,继外膜蛋白、附屑结构蛋白、粘附蛋白以及凝集素等表面蛋白之后,S-层蛋白被作为一种新的表面展示载体成功的在细胞表面展示了一些外源大分子。多聚组氨酸肽是由若干个单位的六聚组氨酸串联而成的短肽。六聚组氨酸能够有效的吸附镍、镉等重金     

苏云金芽胞杆菌CTC菌株的S-层蛋白可以形成伴胞晶体

苏云金芽胞杆菌(Bacillus thuringiensis)CTC菌株产生卵圆形伴胞晶体,晶体蛋白分子量为100kD;透射电子显微镜观察结果表明该菌株有S—层结构,而且在母细胞内可以形成伴胞晶体和S—层的初体结构;其蛋白基因导入苏云金芽胞杆菌无晶体突变株BMB171后,扫描电子显微镜观察结果表明转化子能形成晶体,而其形状与CTC菌株的相同;转化子晶体蛋白的分子量大小也与CTC菌株的相同,为100kD。以上实验结果结合以前晶体蛋白N—末端测序和基因核苦酸序列,表明苏云金芽胞杆菌CTC菌株的S—层蛋白可以形成伴胞晶体。     

类似S-层蛋白的苏云金芽胞杆菌伴胞晶体蛋白基因的克隆

芽胞杆菌CTC菌被鉴定为苏云金芽胞杆菌,鞭毛血清型H2,幕虫亚种;产生卵圆形伴胞晶体,伴胞晶体蛋白为100kD;测定了该蛋白 N－末端序列,该序列与炭疽芽胞杆菌的细胞表面S－层蛋白具92－93％相似性,根据Southern杂交制作了该晶体蛋白基因ctc所在位置的限制性酶切图谱,分别克隆了该基因5’和3’端所在2．9kb XbaI片段和3．1kb Cla I DNA片段,彼此间具0．6kb重叠,通过拼接获得含完整ctc基因的克隆,含该基因的大肠杆菌与表达S－层蛋白的大肠杆菌具相似生长特征,初步表明CTC菌株的伴胞晶体由细胞表面S－层蛋白组成,苏云金芽胞杆菌区别于蜡状芽胞杆菌和炭疽芽胞杆菌的唯一标准是能形成伴胞晶体,由于S－层是细胞表面的结构成分,本文对CTC菌株鉴定为苏云金芽胞杆菌以及伴胞晶体作为苏云金芽胞杆菌鉴别的唯一标准提出了质疑。     

Cloning,production and expression of the bacteriocin enterocin A produced by <Emphasis Type="Italic">Enterococcus faecium</Emphasis> PLBC21 in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Two genes, ctc and ctc2, responsible for surface layer (S-layer) protein synthesis in Bacillus thuringiensis CTC, were mutated and resulted in B. thuringiensis Tr5. To synthesize and express the N-acyl-homoserine lactonase (AHL-lactonase) in the extracellular space of B. thuringiensis, the aiiA _4Q7 gene (an AHL-lactonase gene from B. thuringiensis 4Q7), which confers the ability to inhibit plant soft rot disease in B. thuringiensis 4Q7, was fused with the upstream sequence of the ctc gene, which in turn is essential for S-layer protein secretion and anchoring on the cell surface. The resulting fusion gene, slh-aiiA, was expressed in B. thuringiensis Tr5 to avoid competition for the extracellular space with the native S-layer protein. Our results indicate that B. thuringiensis Tr5 containing the fusion gene slh-aiiA displayed high extracellular AHL-degrading activity. When compared with wild-type B. thuringiensis strains, the ability of the constructed strain to inhibit soft rot disease caused by Erwinia carotovora SCG1 was markedly increased. These findings provide evidence for a significant advance in our ability to inhibit soft rot disease caused by E. carotovora.     

Bacterial SLH domain proteins are non-covalently anchored to the cell surface via a conserved mechanism involving wall polysaccharide pyruvylation

Several bacterial proteins are non-covalently anchored to the cell surface via an S-layer homology (SLH) domain. Previous studies have suggested that this cell surface display mechanism involves a non-covalent interaction between the SLH domain and peptidoglycan-associated polymers. Here we report the characterization of a two-gene operon, csaAB, for cell surface anchoring, in Bacillus anthracis. Its distal open reading frame (csaB) is required for the retention of SLH-containing proteins on the cell wall. Biochemical analysis of cell wall components showed that CsaB was involved in the addition of a pyruvyl group to a peptidoglycan-associated polysaccharide fraction, and that this modification was necessary for binding of the SLH domain. The csaAB operon is present in several bacterial species that synthesize SLH-containing proteins. This observation and the presence of pyruvate in the cell wall of the corresponding bacteria suggest that the mechanism described in this study is widespread among bacteria.     

Production, secretion, and cell surface display of recombinant Sporosarcina ureae S-layer fusion proteins in Bacillus megaterium

Monomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report that Bacillus megaterium is an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA from Sporosarcina ureae ATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N- and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into the Escherichia coli-Bacillus megaterium shuttle vector pHIS1525. After transformation of the respective plasmids into Bacillus megaterium protoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemble in vitro into highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope of Bacillus megaterium, indicating that the cell surface can serve in vivo as a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.     

乳酸乳球菌食品级诱导表达系统的构建及异源蛋白的表达

以α-aga基因为食品级选择标记构建了乳酸乳球菌食品级高效诱导细胞内和细胞壁锚定表达系统,并用这一表达系统表达了铜绿假单胞菌融合外膜蛋白基因OprF/H。首先以pRAF800和pNZ8048构建了含有α-aga、PnisA-MCS-TpepN和θ复制子的乳酸乳球菌食品级细胞内诱导表达载体pRNA48,再以pRNA48和pVE5524为出发载体构建了含有α-aga、PnisA-SPUsp45-nucA-CWAM6-t1t2和θ复制子的乳酸乳球菌细胞壁锚定诱导表达载体pRNV48。然后以食品级载体pRNA48和pRNV48为基础,构建了不含抗生素抗性选择标记的铜绿假单胞菌融合外膜蛋白基因的表达质粒pRNA48-OprF/H和pRNV48-OprF/H。利用nisin进行重组乳酸乳球菌菌株的诱导表达,通过SDS-PAGE和Western blot分析,检测到表达蛋白分别占细胞内可溶蛋白的9.6%和细胞壁锚定蛋白的9.8%,表达产物具有免疫原性,可与含OprF/H的乳球菌以及铜绿假单胞菌发生特异性的凝集反应。     

Surface display of the 20-kDa N-terminal fragment of anthrax protective antigen based on attenuated recombinant <Emphasis Type="Italic">Bacillus anthracis</Emphasis>

Extracellular antigen 1 (EA1), a major component of the Bacillus anthracis surface layer (S-layer), was used as a fusion partner for the expression of heterologous antigen. A recombinant B. anthracis strain was constructed by integrating a translational fusion harboring the DNA fragments encoding the cell wall–targeting domain of the S-layer protein EA1 and the 20-kDa N-terminal fragment of anthrax protective antigen (PA20) into the chromosome. A thermosensitive plasmid expressing Cre recombinase was introduced at a permissive temperature to remove the antibiotic marker. Cre recombinase action at the loxP sites excised the spectinomycin resistance cassette. The final derivative strains were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, Western blot analysis, and immunofluorescence analysis. PA20 was successfully expressed on the S-layer of the recombinant antibiotic marker-free strain. Guinea pigs were immunized with the attenuated recombinant B. anthracis strain, and the bacilli elicited a humoral response to PA20. This antibiotic marker-free strain and the correlative experiment method may have potential applications for the generation of a live attenuated anthrax vaccine.