旋毛虫两个发育时期丝氨酸蛋白酶的结构及功能的比较与分析

目的比较并分析旋毛虫成虫期及新生幼虫期丝氨酸蛋白酶基因和蛋白的结构及功能。方法应用NCBI、EMBL、MEGA、ExPasy、TMHMM、SignalP、PROSITE、Coils、SYFPEITHI等多种在线生物信息学网站和软件,对旋毛虫成虫期和新生幼虫期丝氨酸蛋白酶基因(Zh68和T668)的同源性、系统发育进化状况进行分析,并预测相应蛋白(SP1和SP2)的基本理化性质、跨膜区、motif、结构域、亚细胞定位、二级及三级结构、细胞抗原表位等。结果 Zh68和T668为旋毛虫特有基因,但核苷酸同源性仅为48%,SP1和SP2的氨基酸同源性为27.5%。SP1和SP2均为不稳定的亲水性蛋白,N-末端均有一个信号肽的分泌蛋白;二级及三级结构类似,并预测出两者最佳B及T细胞抗原表位,但SP2有一个跨膜螺旋结构,为膜蛋白。结论旋毛虫成虫期的SP1是非跨膜蛋白,可能参与虫体在肠道寄生及发育、繁殖;新生幼虫期的SP2是跨膜蛋白,可能参与虫体的入侵及迁移。     

人源尖吻蝮蛇毒蛋白抗体单链可变区片段噬菌体文库的初建

目的构建人源尖吻蝮蛇毒蛋白抗体单链可变区片段(scFv)噬菌体文库。方法采用Trizol试剂提取尖吻蝮蛇咬伤后恢复期患者的外周血淋巴细胞的总RNA,用Oligotex誖mRNA Mini Kits纯化获得总mRNA,逆转录合成总cDNA,针对人抗体轻链和重链可变区基因设计一系列特异性引物,以合成的总cDNA为模板,扩增得到抗体轻链和重链可变区基因库,再通过重叠延伸PCR(splicing by overlap extension PCR,SOE-PCR)将轻链和重链拼接得到scFv文库;将scFv基因双酶切后,插入T7噬菌体骨架进行包装,以尖吻蝮蛇蛇毒蛋白抗原进行4轮淘选,建立蛇毒蛋白抗体scFv噬菌体文库,采用噬斑法检测scFv噬菌体文库滴度并进行PCR及测序鉴定。结果成功地将轻链和重链可变区基因库混合拼接得到scFv文库;经4轮淘选,蛇毒蛋白抗体scFv噬菌体文库滴度为6.0×1014PFU/ml,抗体基因在噬菌体中表达的阳性率达50%以上,表达的scFv片段均为轻链和重链的杂合体,可较好的重现可变区的多样性。结论已成功构建了人源尖吻蝮蛇毒蛋白抗体scFv噬菌体文库,为下一步噬菌体文库的克隆构建和筛选奠定了基础。     

抗SARS-CoV-2刺突糖蛋白受体结合域单链抗体的筛选及鉴定

目的 构建抗严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-Co V-2)刺突糖蛋白(spike protein,S)受体结合域(receptor-binding domain,RBD)的单链抗体(single-chain fragment variable,scFv)噬菌体展示文库,筛选特异性scFv,并进行功能鉴定。方法 提取RBD蛋白免疫小鼠的脾细胞m RNA,逆转录为c DNA,以其为模板扩增scFv的重链可变区(hight chain fragment of variable,VH)和轻链可变区(light chain fragment of variable,VL)基因,经过重叠延伸PCR扩增(splicing overlap extension PCR,SOE-PCR)组装为scFv基因片段。将scFv基因片段插入噬菌体载体,构建scFv噬菌体展示文库。经4轮生物淘洗,筛选出与RBD结合能力较强的scFv基因,进行重组表达、纯化和生物活性鉴定。结果 构建的scFv噬菌体文库滴度为6.0...     

从噬菌体表面展示肽库中筛选边缘无浆体膜表面蛋白5单克隆抗体识别的抗原表位

目的从噬菌体表面展示肽库中筛选边缘无浆体膜表面蛋白5(Membrane surface protein5,MSP5)单克隆抗体识别的抗原表位。方法用MSP5单克隆抗体1D8作为靶标,对噬菌体展示随机12肽库进行筛选,通过ELISA和竞争抑制ELISA鉴定筛选克隆的结合特性,并提取阳性克隆的单链DNA,进行测序分析。结果从表面展示随机肽序列的噬菌体文库中筛选到与MSP5单抗1D8特异结合的噬菌体克隆,其一致序列为LING。竞争抑制试验表明,含特异序列的克隆能与MSP5重组蛋白抗原竞争。结论初步确定MSP5单克隆抗体1D8的抗原表位为线性表位,为进一步研究其在边缘无浆体诊断及新型疫苗研制中的作用奠定了基础。     

旋毛虫感染诱导的小鼠乳腺癌MCF-7细胞基因表达的变化

目的构建旋毛虫感染诱导的小鼠乳腺癌MCF-7细胞基因表达变化的cDNA消减文库,探讨旋毛虫感染诱导的MCF-7细胞基因表达的变化。方法复制MCF-7细胞小鼠模型,设对照组和旋毛虫感染组(实验组),采用抑制性消减杂交技术构建旋毛虫感染诱导的MCF-7细胞上调基因表达消减文库,并利用反向Northernblot技术对所获得基因的特异性表达进行验证。结果成功构建了旋毛虫感染诱导的MCF-7细胞上调基因表达消减文库,克隆的目的基因片段大小分布在200～500bp之间,20个阳性克隆的测序结果经BLAST比对,获得差异表达的已知功能基因8个和未知功能基因4个,这些基因在小鼠乳腺癌细胞中均有表达,而在肌肉组织中无表达。结论旋毛虫感染诱导的MCF-7细胞差异表达基因上调,其中RB基因可能与乳腺癌细胞的生长抑制密切相关,为在基因水平研究旋毛虫抗肿瘤机理奠定了基础。     

生物信息学方法筛选华支睾吸虫成虫功能基因——钙调神经磷酸酶B亚基样蛋白

目的本文通过建立华支睾吸虫成虫cDNA文库,筛选其功能基因。方法应用SMART方法构建华支睾吸虫成虫cDNA文库,进行大量EST测序,然后应用生物信息学方法将EST序列与GenBank中登陆的序列进行同源性比对、序列拼接及基因完整性判断;并应用NCBI上的RPSBLAST对所筛选基因的保守域进行搜索比对,利用PredictProtein分析、预测其功能域及二级结构。结果从华支睾吸虫成虫cDNA文库中筛选的钙调神经磷酸酶B亚基样蛋白(CsCBLP)基因,经同源性分析,CsCBLP与褐家鼠钙调神经磷酸酶(CaN)同源性为53%,与尾刺耐格里原虫CaNB同源性为40%,与新小杆线虫属结合蛋白的同源性为51%;保守域的比对及功能域、二级结构的预测显示所筛选的CsCBLP是钙调神经磷酸酶B亚基的的类似物,属于钙结合蛋白家族。结论应用生物信息学方法从华支睾吸虫成虫cDNA文库中筛选出钙调神经磷酸酶B亚基样蛋白基因。     

抗犬瘟热病毒噬菌体抗体库的构建

目的构建抗犬瘟热病毒(canine distemper virus,CDV)T7噬菌体单链抗体(single-chain antibody fragment,scFv)库。方法用灭活CDV经皮下注射免疫犬,共3次,每次间隔2周,第3次免疫后2周采集犬外周血,分离淋巴细胞,提取淋巴细胞总RNA,RT-PCR分别扩增犬IgG重链(heavy chain,VH)和轻链(light chain,VL)基因片段,通过SOE-PCR法将VH和VL用一段linker连接组装成scFv基因,经EcoRⅠ和HindⅢ双酶切后,连接到表达载体T7 Select 10-3b上,用包装蛋白包装后,以大肠埃希菌BLT5403为宿主菌进行增殖,随机挑取50个噬菌斑,进行PCR鉴定,并测定文库滴度。结果扩增的VH和VL基因大小均与预期相符,测序结果经blast比对,扩增的片段与犬IgG的VH和VL序列匹配率达90%以上;所构建的原始文库滴度为3.2×107pfu/ml,扩增后的文库滴度为5.0×108pfu/ml。对随机挑取的50个噬菌斑进行PCR鉴定,重组率为94%。结论成功构建了抗CDV的T7噬菌体抗体库,为犬科动物犬瘟热的防治提供了参考。     

乙肝乙脑麻疹多表位噬菌体疫苗的构建及其免疫原性

目的构建乙肝乙脑麻疹多表位噬菌体疫苗,并检测其免疫原性。方法将PCR方法合成的乙肝乙脑麻疹多表位基因COM插入T7噬菌体基因组中,使其与噬菌体10B蛋白融合,展示在T7噬菌体表面,构建成多表位噬菌体疫苗COM/T7。分别以1010pfu/只和1012pfu/只两种剂量,通过腹腔、皮下和鼻腔3种途径免疫昆明小鼠,ELISA检测小鼠特异性抗体水平。结果重组噬菌体COM/T7经PCR鉴定证明构建正确。乙肝乙脑麻疹多表位噬菌体疫苗能诱导小鼠产生针对各表位的特异性IgG抗体。在3种免疫途径中,腹腔免疫效果最好。免疫剂量与诱导的抗体水平在一定范围内呈正相关。多表位噬菌体疫苗诱导的抗-HBs和抗-JEV水平高于常规疫苗,而抗-MV水平低于常规疫苗。结论构建的乙肝乙脑麻疹多表位噬菌体疫苗具有良好的免疫原性。     

噬菌体展示疫苗的研究进展

噬菌体是感染细菌、真菌、放线菌和螺旋体等微生物的病毒,因其固有的免疫原性、遗传可塑性、稳定性及安全性等优势,使其在疫苗研发中具有独特的潜力。目前,有较多利用其构建疫苗递送平台的研究,主要包括噬菌体展示疫苗、噬菌体DNA疫苗及杂交噬菌体DNA疫苗3种形式,其中研究最为广泛的是噬菌体展示疫苗。噬菌体展示技术是一种新型疫苗制备技术,是以噬菌体为载体,通过将外源多肽或蛋白基因整合至噬菌体基因中,以融合蛋白的形式展示在噬菌体表面的分子生物学技术。本文主要就噬菌体展示疫苗的免疫学基础、展示系统及其在疾病预防应用的研究进展作一综述,以期为噬菌体展示疫苗的研发与应用提供参考。     

仙台病毒Tianjin株噬菌体Fab抗体库的构建及初步筛选

目的构建仙台病毒Tianjin株噬菌体Fab抗体库,并进行初步筛选。方法用仙台病毒Tianjin株免疫小鼠,制备脾细胞悬液,提取小鼠脾细胞总RNA,RT-PCR扩增鼠抗体轻链(κ链)和重链Fd基因,将轻链基因和噬菌粒p3MH经SacⅠ和XbaⅠ双酶切后纯化回收,经T4 DNA连接酶连接,电转化E.coli XL1-Blue,构建轻链库;将重链Fd段基因和轻链库p3MH-κ经SpeⅠ和XhoⅠ双酶切后,纯化回收并连接,电转化E.coli XL1-Blue,获得噬菌体抗体库,计算重组率和抗体库滴度。以仙台病毒Tianjin株重组蛋白HN2为抗原进行初步筛选,制备噬菌体抗体,并进行测序。结果构建的免疫噬菌体抗体库库容为1.18×107,重组率为90%,制备的噬菌体抗体滴度为1011pfu/ml;经初步筛选,噬菌体富集了约63.6倍,获得2株与重组蛋白HN2有结合活性的阳性克隆,经NCBI BLAST进行同源性分析,显示为鼠源性抗体,经IMGT分析,显示轻链基因与Vk9亚群基因的同源性为94.87%,重链基因与VH7亚群基因的同源性为97.85%。结论成功构建了仙台病毒Tianjin株噬菌体Fab抗体库,为该病毒株的诊断、疾病治疗及致病机制等研究奠定了基础。     

旋毛虫成虫与肌幼虫排泄分泌抗原蛋白组分的比较分析

目的比较大理猪源旋毛虫(Trichinela spiralis)成虫与肌幼虫排泄分泌抗原(Excretory-secretory antigens,ES)的蛋白组分差异,并进一步分析其反应原性,为分离筛选出免疫原性和反应原性强的旋毛虫抗原成分奠定基础。方法分别收集和纯化旋毛虫成虫、肌幼虫虫体,制备ES,采用SDS-PAGE分析成虫和肌幼虫ES蛋白成分,Western blot分析其反应原性。结果经SDS-PAGE分析,旋毛虫成虫ES显示17条蛋白条带,相对分子质量范围在120000～14000之间,其中主带6条,相对分子质量分别为120000、64000、43000、40000、35000、33000;旋毛虫肌幼虫ES显示20条蛋白条带,相对分子质量范围在112000～10000之间,其中主带11条,相对分子质量分别为112000、66000、56000、55000、53000、49000、45000、43000、25000、21000、10000。Westernblot分析表明,旋毛虫成虫ES抗原显示7条反应带,相对分子质量分别为43000、40000、35000、27000、19000、18000、14000,其中相对分子质量43000、40000、27000、18000的条带着色明显;旋毛虫肌幼虫ES抗原显示14条反应带,相对分子质量范围在74000～12000之间,其中相对分子质量53000、49000、45000、43000、35000、27000、18000、12000的条带显色明显。结论旋毛虫成虫和肌幼虫ES抗原蛋白组分均复杂,有共同组分,也有不同组分,旋毛虫ES抗原具有较强的反应原性,是旋毛虫病研究的重要候选抗原。     

Selection of an active enzyme by phage display on the basis of the enzyme's catalytic activity in vivo

We have developed a novel phage display method based on catalytic activity for the in vivo selection of an enzyme. To confirm the validity of our method and to demonstrate its potential utility, we used biotin protein ligase (BPL) from Escherichia coli as a model enzyme. We were able to demonstrate the potential value of our method by selective enrichment for the birA gene, which encodes BPL, in a mixed library. The presented method for in vivo selection should allow selection of various enzymes that catalyze modification of peptides or proteins, such as protein ligase, acetylase, kinase, phosphatase, ubiquitinase, and protease (including caspase). The method should be useful in efforts to analyze mechanisms of signal transduction, to find unidentified enzymes encoded by cDNA libraries, and to exploit artificial enzymes.     

Efficient production of antibodies against a mammalian integral membrane protein by phage display

The application of phage display technology to mammalian proteins with multiple transmembrane regions has had limited success due to the difficulty in generating these proteins in sufficient amounts and purity. We report here a method that can be easily and generally applied to sorting of phage display libraries with multispan protein targets solubilized in detergent. A key feature of this approach is the production of biotinylated multispan proteins in virions of a baculovirus vector that allows library panning without prior purification of the target protein. We obtained Fab fragments from a na?ve synthetic antibody phage library that, when engineered into full-length immunoglobulin (Ig)G, specifically bind cells expressing claudin-1, a protein with four transmembrane regions that is used as an entry co-receptor by the hepatitis C virus (HCV). Affinity-matured variants of one of these antibodies efficiently inhibited HCV infection. The use of baculovirus particles as a source of mammalian multispan protein facilitates the application of phage display to this difficult class of proteins.     

人源性抗甲型肝炎病毒Fab噬菌体抗体分子的初步筛选

目的从自建的大容量非免疫Fab噬菌体抗体库中筛选具有抗甲型肝炎病毒(HAV)抗原活性的噬菌体抗体,并进行鉴定。方法采用纯化的甲型肝炎病毒作为包被抗原,对自建的容量为1.26×1011pfu/ml的Fab噬菌体抗体库进行筛选和富集,分别对原始库和筛选后的抗体库进行Phage-ELISA和活性检测,经酶切鉴定和DNA测序分析后进行诱导表达。结果经2轮筛选,抗-HAV抗原的噬菌体抗体得到了明显富集,ELISA检测证实其具有良好的抗甲肝病毒抗原的特异性,抑制率为38.61%。酶切鉴定表明抗体库重组较稳定。SDS-PAGE检测显示Fab抗体蛋白在E.coliDH5α中得到了表达。结论从自建的非免疫Fab噬菌体抗体库中成功获得了抗甲肝病毒噬菌体抗体,所构建的非免疫抗体库可用于人源性抗体的筛选。     

High-Throughput Sequencing of Phage Display Libraries Reveals Parasitic Enrichment of Indel Mutants Caused by Amplification Bias

The combination of phage display technology with high-throughput sequencing enables in-depth analysis of library diversity and selection-driven dynamics. We applied short-read sequencing of the mutagenized region on focused display libraries of two homologous nucleic acid modification eraser proteins—AlkB and FTO—biopanned against methylated DNA. This revealed enriched genotypes with small indels and concomitant doubtful amino acid motifs within the FTO library. Nanopore sequencing of the entire display vector showed additional enrichment of large deletions overlooked by region-specific sequencing, and further impacted the interpretation of the obtained amino acid motifs. We could attribute enrichment of these corrupted clones to amplification bias due to arduous FTO display slowing down host cell growth as well as phage production. This amplification bias appeared to be stronger than affinity-based target selection. Recommendations are provided for proper sequence analysis of phage display data, which can improve motive discovery in libraries of proteins that are difficult to display.     

Rapid isolation of high-affinity protein binding peptides using bacterial display

A robust bacterial display methodology was developed that allows the rapid isolation of peptides that bind to arbitrarily selected targets with high affinity. To demonstrate the utility of this approach, a large library (5 x 10(10) clones) was constructed composed of random 15-mer peptide insertions constrained within a flexible, surface exposed loop of the Escherichia coli outer membrane protein A (OmpA). The library was screened for binding to five unrelated proteins, including targets previously used in phage display selections: human serum albumin, anti-T7 epitope mAb, human C-reactive protein, HIV-1 GP120 and streptavidin. Two to four rounds of enrichment (2-4 days) were sufficient to enrich peptide ligands having high affinity for each of the target proteins. Strong amino acid consensus sequences were apparent for each of the targets tested, with up to seven consensus residues. Isolated peptide ligands remained functional when expressed as insertional fusions within a monomeric fluorescent protein. This bacterial display methodology provides an efficient process for identifying peptide affinity reagents and should be useful in a variety of molecular recognition applications.