天蚕素A-蜂毒素杂合肽用pBV220载体的表达、纯化和活性测定

为提高抗菌肽的表达,在抗菌肽的N端融合了1段酸性小肽以中和表达产物对宿主的毒性;并将融合肽基因同向串连成多拷贝,在大肠杆菌中获得了较高的表达。用化学合成法分别合成了编码天蚕素A(1-8)-蜂毒素(1-10)杂合肽和酸性小肽的DNA片段,首先将其拼接成融合肽的完整基因,然后通过前后接头将融合肽基因连接成两侧具有EcoRI和SalI酶切位点的同向串连的多拷贝基因。将5份拷贝的基因克隆至pBV220表达载体,转化E.coliDH5α,温度诱导得到表达量为35%的融合蛋白。表达产物主要以包涵体形式存在,将包涵体溶解,经Ni²⁺-NTA琼脂糖亲和层析获得纯化的融合蛋白。融合蛋白再经CNBr切割和阳离子交换层析,得到纯化的抗菌肽,经蛋白质N端测序确认序列正确。琼脂糖扩散法和液相测定法证明了纯化的抗菌肽具有抗菌活性。     

BLyS对噬菌体随机12肽库的筛选

用B淋巴细胞刺激因子（BLyS）对噬菌体随机12肽库进行亲和淘洗,3轮筛选后阳性噬菌体得到富集。用ELISA鉴定噬菌体克隆,多个阳性克隆测序后得到了同一个小肽序列(RHKIQLRQNIIT)。将该小肽与GST融合,在大肠杆菌中进行表达及纯化,ELISA实验进一步验证了其具有与BLyS特异结合的活性。该小肽有可能成为其天然受体的拮抗剂。      

抗菌肽Pek Ⅱ基因在大肠杆菌中的融合表达及初步纯化

目的:研究抗茵肽PekⅡ基因在大肠杆菌中的融合表达并初步纯化.方法:根据大肠杆菌密码子的偏好性,人工设计并合成2段核苷酸序列,退火获得PckⅡ基因;将此基因克隆到原核表达载体pGEX-4T-2中,构建成抗茵肽基因PekⅡ融合表达载体pGEX-PekⅡ,转化至大肠杆菌BL21中,用IPTG进行诱导.取超声破碎后的上清经Glutathione SepharoseTM 4亲和层析得到纯化融合蛋白.结果:PCR和测序表明已获得正确PekⅡ编码基因,SDS-PAGE显示29kD处有特异性的蛋白条带出现,纯化得到的GST-PekⅡ经MALDI-TOF MS分析得出其相对分子质量为29553.03.结论:抗菌肽PckⅡ基因在大肠杆菌中的融合表达获得成功,初步纯化得到纯品.     

C19orf18蛋白的表达与纯化及多克隆抗体制备

旨在得到较高浓度和纯度的C19orf18蛋白,再将得到的目的蛋白用于制备其特异性的多克隆抗体。利用PCR技术扩增出C19orf18基因的胞内段和胞外段,中间用柔性肽连接,再将目的片段克隆到p ET28a(+)与p ET32a(+)载体上,通过诱导表达少量的目的蛋白,筛选出表达量较高的载体,再用筛选得到的表达量较高的载体大量表达目的蛋白,通过镍柱纯化得到较纯的C19orf18蛋白。用得到的蛋白免疫新西兰白兔,得到的抗血清先经过Protein A纯化,再进行抗原亲和纯化得到C19orf18蛋白多克隆抗体。结果显示,载体p ET28a-C19orf18蛋白表达量高于p ET32a-C19orf18,经过镍柱纯化后得到了较高浓度与纯度的目的蛋白,利用得到的目的蛋白作为抗原成功制备了其特异性的多克隆抗体。在原核细胞中成功表达了C19orf18重组蛋白,并得到了其特异性多克隆抗体,所制备的多克隆抗体可应用于ELISA、蛋白免疫印迹实验。     

家蝇防御素在大肠杆菌中的表达、纯化与抗体制备

家蝇防御素是从家蝇中克隆得到的1种抗菌肽。为了进一步研究家蝇防御素的功能和制备特异性抗体,采用大肠杆菌表达外源蛋白的方法, 进行了家蝇防御素原核表达的研究。根据克隆到的家蝇防御素基因(Mdde) 的cDNA序列, 设计特异性引物, PCR 扩增成熟肽的cDNA片段, 将成熟肽序列重组到表达载体pGEX 4T 1中, 构建m Mdde/pGEX 4T 1重组表达载体, 在大肠杆菌BL21 中诱导表达, 重组表达的融合蛋白GST Mdde占菌体总蛋白的33 4%。纯化得到GST Mdde后, 再用凝血酶将其从特定位点切开, 得到表达的m Mdde。液体抑菌实验结果初步表明, 表达的融合蛋白GST Mdde对细菌生长有一定的抑制作用。利用纯化的GST Mdde融合蛋白, 制备了抗血清。     

ProNGF在大肠杆菌中的表达、纯化和复性

将含有前导肽的人神经生长因子基因(proNGF)克隆在原核表达载体pET15b中, 转化大肠杆菌BL21(DE3)pLysS, 经IPTG诱导实现了目标融合蛋白的高效表达。SDS-PAGE分析表明表达蛋白占全菌总蛋白的20%左右, 表达蛋白主要以包涵体的形式存在。用6 mol/L的盐酸胍溶解包涵体后, 通过Ni2+-NTA柱纯化, 获得纯化的目标融合蛋白, 电泳谱带扫描分析表明蛋白纯度可达90%以上。Western blotting检测显示, 表达产物有较强的免疫学活性。经肠激酶作用后得到proNGF非融合蛋白, 分子量为27 kD, 100 mL表达菌液可获得13.1 mg proNGF蛋白。用透析复性的方法将目的蛋白重折叠, 复性率为18%, 在重折叠过程中前导肽发挥了一定的积极作用。用PC12细胞进行生物活性鉴定, 结果显示复性后的proNGF蛋白具有良好的生物活性。     

新型冠状病毒S蛋白RBD肽段的原核表达与纯化

目的:利用原核系统对新型冠状病毒S蛋白的受体结合域(RBD)肽段进行克隆、表达和纯化,制备高纯度的RBD肽。方法:在RBD肽段N端加入肠激酶位点序列(DDDDK),对应的核酸序列参照原核系统表达偏好进行全基因合成,构建pET-DsbC-RBD融合表达载体,通过原核表达和亲和纯化获得DsbC-RBD融合蛋白,用肠激酶对融合蛋白进行酶切和二次亲和纯化,获得高纯度新冠病毒S蛋白RBD肽。结果:构建了pET-DsbC-RBD表达载体,融合蛋白DsbC-RBD在大肠杆菌中实现了高效表达,经亲和层析纯化,重组融合蛋白DsbC-RBD相对分子质量为58×103,纯度约90%。用肠激酶对融合蛋白进行酶切,酶切效率近100%,通过二次亲和纯化获得了RBD肽,相对分子质量为25×103,纯度92%以上。结论:利用原核表达系统获得可溶性DsbC-RBD融合蛋白,采用亲和纯化和肠激酶酶切联用的方法制备获得高纯度的新冠病毒S蛋白RBD肽,为后续抗体制备和抗病毒药物筛选奠定了基础。     

ProNGF在大肠杆菌中的表达、纯化和复性

将含有前导肽的人神经生长因子基因(proNGF)克隆在原核表达载体pET15b中, 转化大肠杆菌BL21(DE3)pLysS, 经IPTG诱导实现了目标融合蛋白的高效表达。SDS-PAGE分析表明表达蛋白占全菌总蛋白的20%左右, 表达蛋白主要以包涵体的形式存在。用6 mol/L的盐酸胍溶解包涵体后, 通过Ni2+-NTA柱纯化, 获得纯化的目标融合蛋白, 电泳谱带扫描分析表明蛋白纯度可达90%以上。Western blotting检测显示, 表达产物有较强的免疫学活性。经肠激酶作用后得到proNGF非融合蛋白, 分子量为27 kD, 100 mL表达菌液可获得13.1 mg proNGF蛋白。用透析复性的方法将目的蛋白重折叠, 复性率为18%, 在重折叠过程中前导肽发挥了一定的积极作用。用PC12细胞进行生物活性鉴定, 结果显示复性后的proNGF蛋白具有良好的生物活性。     

骨桥蛋白RGD黏附序列多拷贝短肽的表达、纯化及生物学活性测定

目的:用大肠杆菌表达骨桥蛋白RGD黏附序列6拷贝短肽,经分离纯化后检测其生物学活性.方法:运用基因重组技术,将骨桥蛋白RGD黏附序列的核酸片段首尾相连,与携带GST编码序列的原核表达载体连接构建融合蛋白表达质粒pGEX-3X-RGD.将重组质粒转化宿主菌后,对诱导融合蛋白表达的条件进行优化.表达产物GST-RGD经谷胱甘肽-亲和层析纯化后,分别检测其对骨桥蛋白诱导的血管平滑肌细胞黏附和迁移的影响.结果:所构建的含有6个拷贝短肽的GST-RGD融合蛋白可在大肠杆菌中以包含体的形式进行表达.用十二烷基肌氨酸钠变性溶解包含体及透析复性后,经亲和层析可得到高纯度的GST-RGD(6)融合蛋白.GST-RGD(6)融合蛋白能特异性的抑制骨桥蛋白诱导的血管平滑肌细胞的黏附和迁移.结论:骨桥蛋白RGD黏附序列6拷贝短肽可在大肠杆菌中高效表达,纯化的GST-RGD融合蛋白具有抑制血管平滑肌细胞黏附和迁移的活性.     

生物活性肽Lunasin 的原核表达和分离纯化

目的:利用基因工程的方法在大肠杆菌中表达并纯化生物活性肽Lunasin。方法:将合成的Lunasin基因插入原核表达载体pET-32a(+)的多克隆位点Nde I和Xho I之间,然后将重组载体转化入大肠杆菌BL21(DE3)中,利用IPTG诱导表达蛋白,经SDS-PAGE和Western Blot鉴定蛋白的表达。然后利用亲和层析技术将含有6×His标签的蛋白分离纯化、脱盐、冻干。结果:①鉴定结果表明在6kDa位置出现目的条带Lunasin重组蛋白。②亲和层析在100mM咪唑时得到了洗脱的重组蛋白。结论:在大肠杆菌BL21(DE3)中成功表达并且纯化出了生物活性肽Lunasin。     

死亡素与泛素在大肠杆菌中的高效融合表达

死亡素是由21个氨基酸残基组成的广谱抗菌肽。为了高效表达可溶性的死亡素,本研究利用递归式PCR（recursive PCR, rPCR）扩增了死亡素基因thanatin,并将其和家蝇Musca domestica泛素基因ubiquitin构成嵌合基因,克隆到表达载体pET-32a,再与硫氧还蛋白融合后构建表达载体pET-TRX-UBI-THA。将酶切和测序鉴定正确的质粒转化表达宿主菌BL21,经0.6 mmol/L IPTG诱导,TRX-UBI-THA融合蛋白得到了高效可溶性表达。SDS-PAGE和Western blot检测结果表明融合蛋白的分子量为28.9 kD,与预期的结果一致,表达量占菌体总蛋白的46％。Western blot分析结果显示融合蛋白能与Ni-NTA鏊合物特异性的结合,表明在融合蛋白的N-端带有6×His标签。利用C-端带有6×His标签的泛素C-端水解酶对融合蛋白进行切割,切割产物经Ni²⁺-NTA亲和柱和HPLC纯化（纯化量为5.4 mg/L）,Tricince-SDS-PAGE电泳得到单一的泛素蛋白条带。电喷雾质谱(ESI-MS)分析表明,纯化的泛素分子量为2.57 kD,与通过氨基酸预测的分子量完全一致。利用琼脂孔穴扩散法对泛素活性进行检测,结果显示纯化的泛素对大肠杆菌K12D31和金黄色葡萄球菌Staphylococcus aureus具有较强的活性抑制。本研究表明,利用泛素融合技术可以高效表达可溶性的死亡素。     

家蚕抗菌肽-死亡素杂合肽基因在大肠杆菌中的克隆与表达

近年来的研究发现 ,抗菌蛋白在生物体非专一性防御系统有着重要的作用 ,已有数十种具有抗菌活性的多肽被分离 ,这些多肽可大致分为 3类 ,即含分子内二硫桥的抗菌肽 ;具有双亲α 螺旋结构的抗菌肽 ;以及富含某种氨基酸残基的抗菌肽[1 ] ,一般来说 ,这些抗菌肽具有分子量小 ,稳定性好 ,无细胞毒性 ,抗菌谱广等特点。多种抗菌肽的一级结构和二级结构已经确定[2 ] ,但作用机理仍不明了。一般认为可能存在两种作用模式 ,即 1)通过肽 脂膜相关作用杀菌 ;2 )通过受体介导的识别过程起作用[1 ] 。ＣｅｃｒｏｐｉｎＢ是一种较早从家蚕中分离得到 ,由 …     

Expression of antimicrobial peptides thanatin(S) in transgenic Arabidopsis enhanced resistance to phytopathogenic fungi and bacteria

Thanatin(S) is an analog of thanatin, an insect antimicrobial peptide possessing strong and broad spectrum of antimicrobial activity. In order to investigate if the thanatin could be used in engineering transgenic plants for increased resistance against phytopathogens, the synthetic thanatin(S) was introduced into Arabidopsis thaliana plants. To increase the expression level of thanatin(S) in plants, the coding sequence was optimized by plant-preference codon. To avoid cellular protease degradation, signal peptide of rice Cht1 was fused to N terminal of thanatin(S) for secreting the expressed thanatin(S) into intercellular spaces. To evaluate the application value of thanatin(S) in plant disease control, the synthesized coding sequence of Cht1 signal peptide (Cht1SP)-thanatin(S) was ligated to plant gateway destination binary vectors pGWB11 (with FLAG tag). Meanwhile, in order to observe the subcellular localization of Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP, the sequences of Cht1SP-thanatin(S) and thanatin(S) were respectively linked to pGWB5 (with GFP tag). The constructs were transformed into Arabidopsis ecotype Col-0 and mutant pad4-1 via Agrobacterium-mediated transformation. The transformants with Cht1SP-thanatin(S)-FLAG fusion gene were analyzed by genomic PCR, real-time PCR, and western blots and the transgenic Arabidopsis plants introduced respectively Cht1SP-thanatin(S)-GFP and thanatin(S)-GFP were observed by confocal microscopy. Transgenic plants expressing Cht1SP-thanatin(S)-FLAG fusion protein showed antifungal activity against Botrytis cinerea and powdery mildew, as well as antibacterial activity against Pseudomonas syringae pv. tomato. And the results from confocal observation showed that the GFP signal from Cht1SP-thanatin(S)-GFP transgenic Arabidopsis plants occurred mainly in intercellular space, while that from thanatin(S)-GFP transgenic plants was mainly detected in the cytoplasm and that from empty vector transgenic plants was distributed uniformly throughout the cell, demonstrating that Cht1 signal peptide functioned. In addition, thanatin(S) and thanatin(S)-FLAG chemically synthesized have both in vitro antimicrobial activities against P. syringae pv. tomato and B. cinerea. So, thanatin(S) is an ideal candidate AMPs for the construction of transgenic crops endowed with a broad-spectrum resistance to phytopathogens and the strategy is feasible to link a signal peptide to the target gene.     

Design,recombinant expression,and antibacterial activity of a novel hybrid magainin–thanatin antimicrobial peptide

Antimicrobial peptides are small molecule polypeptides with biological activity, which can avoid the drug resistance. Magainin and thanatin are antimicrobial peptides with a broad spectrum of inhibitory microbes, and the core sequence of magainin is linked to a core sequence of thanatin. Here, the hybrid magainin–thanatin (MT) antimicrobial peptide was designed through bioinformatics analysis. The recombinant MT antimicrobial peptide was successfully expressed and purified in Escherichia coli BL21 (DE3). The molecular weight of the hybrid MT antimicrobial peptide was about 3.35?kDa. Moreover, the target protein indeed has an inhibitory effect on Staphylococcus aureus, E. coli DH5α, and Bacillus subtilis, with the minimum inhibitory concentrations 16.5, 20, and 9?μM, respectively. The rational designed hybrid MT antimicrobial peptide will hopefully provide large-scale fermentable antimicrobial peptides in the industrial production in the future.     

High-Level Expression and Novel Purification Strategy of Recombinant Thanatin Analog in Escherichia coli

Recombinant thanatin analog (TH1) is a cationic 20-amino-acid antibacterial peptide with a conserved cysteine disulfide bond. It exhibits a broad antibacterial spectrum. Different strategies have been developed to produce small antibacterial peptides using recombinant techniques. To date, no efforts to obtain large quantities of active recombinant TH1 have been reported. This study describes the synthesis of TH1 gene, the heterologous fusion expression of the peptide in Escherichia coli, and the bioactive assay of released TH1. By constructing the expression plasmid (pET32a-TH1), high yields of soluble TH1 fusion protein (0.416 g/L) can be obtained in E. coli. Further optimization studies have been carried out to increase the expression of TH1 in different culture conditions, with the final amount of pure TH1 being 13.2 mg/L. The results show that the expression system provides a simple and reliable strategy for generating large quantities of TH1 by soluble fusion expression in E. coli.     

Recombinant expression, purification, and antimicrobial activity of a novel hybrid antimicrobial peptide LFT33

With great therapeutic potential against antibiotic-resistant bacteria, viruses, and even parasites, antimicrobial peptides (AMPs) have received increased interest as pharmaceutical agents in recent years. It is a worthy yet challenging work to carry out the implement and improvement of AMPs production using bioengineering techniques. In the present study, a novel hybrid peptide LFT33 was designed derived from LfcinB and thanatin. The cDNA fragment encoding LFT33 with preferred codons of Escherichia coli was chemically synthesized and ligated into the vector pET32a(+) to express the LFT33 fusion protein. The fusion protein was successfully expressed in soluble form in E. coli induced under optimized conditions. After purification by affinity chromatography, the fusion protein was cleaved successfully by enterokinase and released the peptide LFT33. About 0.5?mg of the recombinant LFT33 was obtained by reversed-phase high performance liquid chromatography from 1?l of culture medium. Mass spectrometry analysis of the purified recombinant LFT33 demonstrated that the molecular weight perfectly matched the calculated mass (4,195?Da). The recombinant peptide LFT33 caused an increase in antimicrobial activity (IC(50)?=?16-64?μg/ml) against given strains and did not show hemolytic activity for human erythrocytes. The results indicated that the hybrid peptide LFT33 could serve as a promising candidate for pharmaceutical agents.     

重组人MBD4蛋白在大肠杆菌中的表达、纯化及活性分析

为获得重组人MBD4蛋白,将编码MBD4的开放式阅读框（ORF）插入原核表达载体pGEX6P1 GST基因下游的多克隆位点（MCS）.将获得的表达质粒转化入大肠杆菌BL21(DE3) 菌株扩大培养并用IPTG诱导融合蛋白的表达.用谷胱甘肽琼脂糖凝胶 4B亲和介质从菌体裂解液中纯化了GST-MBD4融合蛋白.经过Prescision protease专一性裂解成功去除了融合蛋白上的GST标签.通过Mono Q阴离子交换层析获得了纯度达94%以上的MBD4蛋白,该蛋白具有甲基化DNA结合和糖苷酶生物活性.     

猪IL-18在毕赤酵母中的表达及活性分析

采用PCR方法从pGEM-IL-18重组质粒中扩增出IL-18基因并构建真核融合表达载体pPIC9K-IL-18,电激法转化入毕赤酵母GS115,采用G418抗性梯度法筛选得到多拷贝重组菌株,甲醇诱导表达,应用SDS-PAGE分析重组蛋白的表达情况,并将表达蛋白用凝胶层析柱纯化后,用MTT法检测其生物学活性。实验结果表明重组的GS115酵母菌株可表达分泌pIL-18,其表达在72h时达高峰,分泌量可达160mg/L,纯化的重组pIL-18蛋白具有显著的促进淋巴细胞增殖的活性,说明本试验已在毕赤酵母中在国内首次成功表达了具有生物学活性的pIL-18。     

霍乱毒素B亚单位基因（CtxB）的克隆及其表达

从霍乱弧菌中抽提基因组ＤＮＡ,用ＰＣＥＲ方法获取霍乱毒素Ｂ亚单位基因（ＣｔｘＢ）。序列分析结果表明,ＣｔｘＢ基因编码１２４个氨基酸,其中编码６２位Ｔｈｒ的密码子与文献报道有差异。将ＣｔｘＢ基因插入质粒ｐＧＥＸ－４Ｔ－２,构建ｐＧＥＸ－ＣＴＸＢ表达质粒,转化大肠相菌ＢＬ２１（ＤＥ３０,筛选表达菌株ＣＴＸＢ／ＢＬ２１。工程株经ＩＰＴＧ诱导表达,可产生大量的表达蛋白,经ＳＤＳ－ＰＡＧＥ分析,融合蛋白分子