基于CRISPR/Cas9系统对乳酸乳球菌进行绿色荧光蛋白标记

乳酸乳球菌是治疗剂在体内运送的良好载体,研究其在体内的真实运送情况需对其进行标记。该实验利用CRISPR/Cas9系统对乳酸乳球菌(Lactococcus lactis)NZ9000进行增强型绿色荧光蛋白(enhanced green fluorescent protein, eGFP)标记,用于研究乳酸乳球菌在体内的运送,评价其作为益生菌的功能。基于该实验室已构建的乳酸乳球菌CRISPR/Cas9编辑质粒pLL25构建重组质粒pYSH,其上携带eGFP及同源臂,电转入乳酸乳球菌NZ9000感受态细胞中,将基因组中的乳酸脱氢酶基因(ldh)替换为绿色荧光蛋白基因,从而使Lactococcus lactis NZ9000获得标记,表达绿色荧光蛋白。对绿色荧光标记的Lactococcus lactis NZ9000突变株,酶标仪定量分析eGFP的表达强度。荧光强度定量分析结果表明,在乳酸乳球菌不同生长阶段,eGFP基因均能稳定表达。     

基于pyrF的乳酸乳球菌食品级表达载体的构建

基于pyrF筛选标记和来源于乳酸乳球菌(Lactococcus lactis,L. lactis)的基因组DNA为表达元件,构建L. lactis食品级表达载体,用于食品和药用多肽的表达和生产。首先,利用NZ3900 pyrF基因列构建同源重组突变盒,构建NZ3900ΔpyrF突变株;然后,分别以来源于L. lactis的repA和repC基因为复制元件、pyrF基因为筛选标记、P₃₂和P₈为启动子、以及Tusp₄₅和TpepN为终止子,构建食品级表达质粒pLD;最后以绿色荧光蛋白ZsGreen为报告基因,验证ZsGreen在NZ3900ΔpyrF突变株的表达及pLD-ZsG的遗传稳定性。实验结果表明,原养型ZsGreen阳性转化子可在普通Elliker培养基中正常生长,在荧光显微镜下观察到明显的绿色荧光信号;此外,PCR和Western blotting也证实ZsGreen能在NZ3900中表达且能稳定传代至30代,说明pLD食品级表达载体构建成功且使得外源蛋白在L. lactis中稳定表达。综上所述,基于pyrF营养...     

乳源血管紧张素转移酶抑制肽在乳酸乳球菌中的表达

在乳酸乳球菌中表达乳源血管紧张素转移酶抑制肽。选取了4种不同的来源于牛酪蛋白的血管紧张素转移酶(ACE)抑制肽,为了确保能够在人体消化液作用下正常发挥它们的ACE抑制活性,4种短肽以串联多肽(TP)的形式进行表达,并在各短肽单体间引入了人体内主要消化酶的酶切位点。根据TP的氨基酸序列和乳酸乳球菌的偏爱密码子,人工合成TP基因。然后将TP基因和绿色荧光蛋白(GFP)基因串联于载体pSEC-E7,从而构建了pSEC-TP:GFP质粒,实现了2种蛋白在乳酸乳球菌中的共表达。经电击转化,将该重组质粒转入乳酸乳球菌NZ9000中,获得重组菌株NZ9000(pSEC-TP:GFP)。用Nisin诱导TP:GFP蛋白表达。RT-PCR、激光共聚焦扫描显微镜和SDS-PAGE鉴定表达产物。RT-PCR结果表明,TP:GFP蛋白在RNA水平表达成功;SDS-PAGE表明目标产物是35 ku的条带。在乳酸乳球菌中实现了乳源血管紧张素转移酶抑制肽的表达。     

具有溶栓活性的重组乳酸乳球菌的构建

利用PCR方法从纳豆芽孢杆菌(Bacillus subtilis subsp.natto)基因组DNA中扩增纳豆激酶原基因,构建了纳豆激酶原基因的表达载体pFY002,转化Lactococcus lactis NZ9000,得到活性表达纳豆激酶的重组菌Lactococcus lactis NZ9000/pFY002。利用纤维蛋白平板法检测其溶栓活性,并对诱导剂乳链菌肽(NisinA)的浓度、温度、诱导时间对重组菌产酶的影响进行探究。     

乳酸乳球菌两组分食品级NICE系统载体的构建

乳酸菌食品级NICE系统是目前较理想的可控制蛋白质生产的食品级诱导表达系统。本实验构建的食品级表达载体pRNA48含α-aga食品级选择标记、θ型复制子和nisA启动子，与宿主菌L．hctis NZ9000共同构成乳酸乳球菌食品级NICE系统，可用于目的基因在乳酸乳球菌中高效诱导表达。     

大豆锰超氧化物歧化酶基因在乳酸乳球菌中的分泌表达

为使大豆锰超氧化物歧化酶（Mn superoxide dismutase,MnSOD）在乳酸乳球菌中高效表达,将克隆的MnSOD基因开放阅读框序列分别重组到质粒pNZ8149和经改造的质粒pNZS上,表达载体pNZ-SOD和分泌表达载体pNZS-SOD,将两者分别以电穿孔法转化乳酸乳球菌L. lactis NZ3900,经Elliker琼脂板筛选、聚合酶链式反应（polymerase chain reaction,PCR）、酶切鉴定正确后,获得的两重组菌株加入Nisin进行诱导表达,对L. lactisNZ3900/pNZ-SOD和L. lactis NZ3900/pNZS-SOD的表达产物进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodiumdodecyl sulfate - polyacrylamide gel electrophoresis,SDS-PAGE）和酶活性对比分析。结果显示：L. lactis NZ3900/pNZS-SOD菌株表达SOD总活性约是L. lactis NZ3900/pNZ-SOD菌株的1.6 倍,是L. lactis NZ3900/pNZ8149的13.5倍,且可将表达的约2/3的SOD分泌到胞外,表明经改造的乳酸菌分泌表达系统L. lactis NZ3900/pNZS能够分泌表达SOD,并增强SOD的表达。     

过量表达purC基因对Lactococcus lactis NZ9000酸胁迫抗性的影响

通过在乳酸乳球菌Lactococcus lactis NZ9000中过量表达嘌呤代谢途径中编码磷酸核糖基氨基咪唑-琥珀酰胺合酶的pur C基因,测定了重组菌株在酸胁迫条件下的存活率,经p H 4. 0胁迫培养4 h后,重组菌株的存活率为对照菌株的83. 2倍。采用ATP测定试剂盒和高效液相色谱分别考察菌株胞内ATP和氨基酸含量。结果表明,重组菌株在酸胁迫条件下维持了更高的胞内ATP和氨基酸(天冬氨酸、苏氨酸、谷氨酸和γ-氨基丁酸)含量,分别通过为细胞提供能量和消耗胞内质子的方式,帮助乳酸乳球菌抵御酸胁迫。研究发现,在乳酸乳球菌中过表达pur C基因能够明显提高菌株的酸胁迫抗性,同时为进一步通过改造嘌呤代谢途径提高乳酸菌酸胁迫耐受性提供了新的思路。     

TGEV和PEDV融合S蛋白在乳酸乳球菌食品级细胞壁锚定高效诱导表达与免疫原性分析

近年来猪传染性胃肠炎和猪流行性腹泻在我国广泛流行,给养殖业带来了巨大的经济损失。目前对此两种疾病的防治手段还停留在使用常规疫苗。现有的疫苗免疫效果不够理想,急需寻求一种安全高效的防治手段。本文首先构建了猪传染性胃肠炎病毒(TGEV)和猪流行性腹泻病毒(PEDV)融合S基因,以乳酸乳球菌食品级细胞壁锚定高效诱导表达载体p RNV48为基础,构建了TGEV和PEDV融合S基因的表达质粒p RNV48-TPs。将得到的重组质粒用电穿孔方法转入乳酸乳球菌NZ9000感受态细胞中,获得了重组菌乳酸乳球菌Z9000/p RNV48-TPs。再利用nisin对重组菌进行诱导,提取细胞壁蛋白后采用聚丙烯酰胺凝胶电泳(SDS-PAGE)分析目的蛋白的表达情况,然后分别对兔子进行注射免疫和口服免疫,并用Western Blot分析。通过乳酸乳球菌食品级细胞壁锚定高效诱导表达载体,诱导表达TGEV与PEDV融合S蛋白产生中和抗体,以期探讨口服疫苗的免疫反应机理,为由TGEV与PEDV研发的安全、高效的疫苗奠定良好的基础。     

棘孢曲霉β-葡萄糖苷酶的乳酸乳球菌表达

构建棘孢曲霉β-葡萄糖苷酶食品级分泌表达载体并在乳酸乳球菌MG1363(L.lactis MG1363)中实现表达。通过PCR扩增L.lactis MG1363基因组中分泌信号肽Usp45、质粒pLEB590的乳链菌肽(Nisin)抗性基因NisI和质粒pPIC9k-Abgl的棘孢曲霉β-葡萄糖苷酶基因Abgl,PCR方法连接后获得片段Usp45-Abgl-NisI,将其克隆到质粒pMD19中,CaCl2法转化到E.coli DH5α,测序鉴定后将该片段连接到大肠杆菌-乳酸菌穿梭质粒pMG36e中并转化到E.coli XL1-Blue,得重组子E.coli XL1-Blue/pMG36e-Usp45-Abgl-NisI。通过PCR方法敲除质粒pMG36e-Usp45-Abgl-NisI中红霉素抗性基因以构建食品级分泌载体pMG36N-Usp45-Abgl-NisI,将其电转到L.lactis MG1363中。转化子E.coli XL1-Blue/pMG36e-Usp45-Abgl-NisI能将β-葡萄糖苷酶分泌到胞外使七叶苷平板显色,L.lactis MG1363/pMG36N-Usp45-Abgl-NisI能在20 IU/mL Nisin上生长,经RT-PCR验证β-葡萄糖苷酶在乳酸乳球菌中实现表达。表明分泌型表达载体构建成功,为其在乳酸乳球菌中实现食品级活性表达奠定基础。     

cA-GFP融合蛋白在大肠杆菌中的高效功能性表达

为了实现乳酸乳球菌锚定蛋白cA在大肠杆菌中可溶表达以及直观检测其生物学活性,以乳酸乳球菌MG1363为模板,扩增N-乙酰葡萄糖胺糖苷酶AcmA基因,将其C末端序列与GFP基因融合,连接至大肠杆菌表达载体pET28a,构建重组质粒pET28a-cA-GFP,转化至大肠杆菌表达菌株BL21(DE3),通过低温诱导表达重组蛋白cA-GFP。工程菌超声破碎后的上清液与经热酸处理的乳酸乳球菌常温孵育,经SDS-PAGE和荧光显微镜检测。结果表明,工程菌可以表达分子量约53ku的目的融合蛋白质,与预期大小相符。cA-GFP通过锚定蛋白cA的引导回向锚定,成功将GFP展示在乳酸乳球菌表面,目的蛋白cA-GFP在乳酸乳球菌表面展示量为121mg/g干重菌体。GFP锚定至乳酸乳球菌后于4℃保存,连续6d测定其荧光强度,荧光强度仍可达82.2%,证明其稳定性较好。成功获得了具有生物学功能的cA-GFP可溶性蛋白,为进一步展示功能性外源蛋白奠定了坚实的基础。     

外源添加亮氨酸提高乳酸乳球菌酸胁迫抗性

在乳酸乳球菌生长过程中外源添加浓度为10mmol/L的亮氨酸,可有效提高乳酸乳球菌在酸胁迫环境下的酸耐受性。在酸性环境中(pH5.0),添加亮氨酸的菌株的生物量为对照菌株的1.24倍;经过酸胁迫(pH4.0)5h后,添加亮氨酸菌株的存活率是对照菌株的28.5倍。进一步的研究表明,亮氨酸的添加可提高胞内NH4+浓度,有效的维持酸胁迫环境下胞内pH(pHin)的稳定,并有效维持乳酸脱氢酶(LDH)的活性,从而有效提高了乳酸乳球菌对酸胁迫的抵御能力。     

表达外源谷氨酸脱羧酶基因对重组乳酸乳球菌胁迫抗性的影响

通过在乳酸乳球菌（Lactobacillus lactis）NZ9000外源添加一定浓度的γ-氨基丁酸（gamma-aminobutyric acid,GABA）和表达可以合成GABA的谷氨酸脱羧酶（glutamic acid decarboxylase,GAD）两种方式,研究L. lactis NZ9000在酸胁迫和冷冻胁迫下的表现。结果表明：外源添加GABA的手段对于L. lactis NZ9000在酸胁迫和冷冻胁迫下的生长没有明显帮助。与之相反,通过逆转录-聚合酶链式反应克隆茶树中的CsGAD,构建pNZ8148-CsGAD表达载体,可获得高效表达GAD的基因工程重组菌L.?lactis?NZ9000/pNZ8148-CsGAD。经2?ng/mL乳酸链球菌素（Nisin）诱导8?h后,高效液相色谱检测表明重组菌合成GABA能力提高5?倍以上。生长曲线测定结果表明,正常培养条件下,重组菌到达对数生长平台期的时间较对照菌延长,到达稳定期的最大生物量（OD600?nm）提高至对照菌的1.5?倍;在pH?4.0的酸性培养条件下,对照菌基本不生长,而重组菌仍保持一定的生长力。低温胁迫实验结果表明,培养至稳定期后期的乳酸菌抗冻能力和存活率均要高于对数期中期和稳定期前期2?个培养阶段,且不同培养阶段中,重组菌的存活率均高于对照菌1～2?个数量级。综上所述,重组的L.?lactis?NZ9000/pNZ8148-CsGAD能够通过食品级异源表达CsGAD产生的GABA显著提高乳酸菌抗酸和抗冷冻胁迫的能力。本研究为探索研究乳酸菌抗胁迫机理、改良乳酸菌生长状态以利于其工业化用途的扩展提供了参考。     

Single-plasmid systems based on CRISPR-Cas9 for gene editing in Lactococcus lactis

Lactococcus lactis is a food-grade lactic acid bacterial species that is widely used in food and medical industries. Due to its relatively small genome and simple metabolism, L. lactis is commonly engineered to produce large quantities of recombinant proteins. The most common single-gene knockout strategy in L. lactis involves RecA-dependent homologous double-crossover recombination, which is relatively time-consuming and laborious. In this study, a precise and efficient genome-editing plasmid for L. lactis NZ9000 genome engineering, pLL, was established based on clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology. By studying the effects of different single guide RNA (sgRNA) promoters, the efficiency of gene deletion was optimized. For LLNZ_02045 (ldh), gene deletion efficiency of up to 50% was achieved. Effective sequential gene deletion of LLNZ_11240 (upp) and LLNZ_04580 (upp1) was also demonstrated using this tool. Additionally, the gene that encodes for uracil phosphoribosyltransferase was identified using this system. Similar robust gene deletion efficiencies of sgRNA that targeted different regions of a single gene suggested that gene deletion was not affected by the location of sgRNA binding. Thus, our study established a new gene-editing tool that may allow further investigation and understanding of the L. lactis NZ9000 genome.     

Chimeras of mature pediocin PA-1 fused to the signal peptide of enterocin P permits the cloning, production, and expression of pediocin PA-1 in Lactococcus lactis

Chimeras of pediocin PA-1 (PedA-1), a bacteriocin produced by Pediococcus acidilactici PLBH9, fused to the signal peptide of enterocin P (EntP), a sec-dependent bacteriocin produced by Enterococcus faecium P13, permitted the production of PedA-1 in Lactococcus lactis. Chimeric genes encoding the EntP signal peptide (SP(entP)) fused to mature PedA-1 (pedA), with or without its immunity gene (pedB), were cloned into the expression vector pMG36c to generate the recombinant plasmids pMPP9 (SP(entP):pedA) and pMPP14i (SP(entP):pedA + pedB). Transformation of competent L. lactis subsp. lactis IL1403, L. lactis subsp. cremoris NZ9000, and L. lactis subsp. lactis DPC5598 with the recombinant plasmids has permitted the detection and quantitation of PedA-1 and the coproduction of nisin A and PedA-1 in supernatants of producer cells with specific anti-PedA-1 antibodies and a noncompetitive indirect enzyme-linked immunosorbent assay. Recombinant L. lactis hosts carrying pMPP9 or pMPP14i displayed antimicrobial activity, suggesting that mature PedA-1 fused to SP(EntP) is the minimum requirement for the synthesis, processing, and secretion of biologically active PedA-1 in L. lactis. However, the production and antimicrobial activity of the PedA-1 produced by L. lactis was lower than that produced by the P. acidilactici control strains.     

Monellin-EGFP 融合蛋白在毕赤酵母中的表达

根据已报道的Monellin甜蛋白氨基酸序列,结合毕赤酵母与桑树密码子的偏好性,人工设计合成单链monellin基因,并与增强型绿色荧光蛋白EGFP基因连接,构建融合表达载体。采用电击转化法成功将表达载体导入毕赤酵母GS115中,并获得高效表达的重组子pPIC9K-M-E。经PCR检测,SDS-PAGE和Western Blot杂交证实已表达出融合蛋白,该蛋白经纯化与盲测实验,结果显示其甜度约为标准蔗糖的500倍。     

CRISPR/Cas9介导烟草多基因编辑体系的应用

CRISPR/Cas9是一种新型的基因组定向编辑技术,近年来在烟草基因组的定向编辑中得到了广泛应用。CRISPR/Cas9介导的多基因编辑技术在许多物种中表现出很大的潜力,为了探索CRISPR/Cas9介导烟草多基因编辑的技术体系,本文针对烟草PCS1、HMA2、eIF4E、TOM3、TOM1 5个不同性状相关的基因构建了多靶点敲除的CRISPR/Cas9系统,并借助农杆菌介导的遗传转化技术转化烟草品种K326。对阳性植株的筛选、靶位点基因组的PCR扩增与测序分析表明,构建的CRISPR/Cas9多基因编辑系统成功转化到烟草中,能够同时靶向突变5个基因。5个基因同时突变的检出率为53.8%,单基因的突变检出率在76.9%与92.3%之间。进一步的脱靶检测分析显示,在所预测脱靶的候选位点上均未发生脱靶现象。本文构建的CRISPR/Cas9多基因编辑系统可将多个基因进行有效突变,为烟草基因功能研究和基于CRISPR/Cas9技术的多性状改良奠定了基础。      

Expression of milk-derived angiotensin I-converting enzyme-inhibitory peptides in Lactococcus lactis

Angiotensin I-converting enzyme (ACE) plays a key role in the regulation of blood pressure. Currently, most single or tandem repeats of ACE-inhibitory (ACE-I) peptides have been expressed in Escherichia coli. However, in this study, a food-grade system was constructed using Lactococcus lactis (L. lactis) to simultaneously express four different milk-derived ACE-I peptides with antihypertensive activity. Mixed peptides (MPs) fused with green fluorescent protein (GFP) and eight histidines were synthesized. To ensure potent ACE inhibition by the MPs in human digestive juice, pepsin and trypsin cleavage sites were introduced among the four ACE-I peptides. The MP fusion gene was inserted into expression vector pSEC-E7 with nisin induction and expressed in L. lactis NZ9000, then purified by affinity chromatography. The transformants containing pSEC-MP:GFP were identified based on green fluorescence using Leica laser scanning confocal microscopy. The target proteins were detected by SDS-PAGE analysis and displayed obvious immunogenicity by western blot. After hydrolysis with digestive enzymes, the IC₅₀ of the MPs was 118.63 μM. These results suggested that multiple milk-derived ACE-I peptides with antihypertensive properties could be produced using a food-grade lactococcal expression system.