人源抗狂犬病毒单链二硫键稳定抗体的重组设计与表达

以人源抗狂犬病毒糖蛋白母本单链抗体ScFv为模板,利用PCR点突变分别在重链FR可变区VH(44)和轻链FR可变区VL(100)分别引入一个半胱氨酸,成功构建了重组单链二硫键稳定抗体基因。连接pET22b( )载体,转化入E.coli BL21(DE3)得到工程菌,IPTG诱导表达。体外复性并经Ni-NTA亲和层析对目的蛋白ScdsFv进行纯化;利用荧光抗体实验和ELISA检测抗体活性及稳定性。结果表明重组ScdsFv蛋白实现了原核高效表达,通过体外复性和Ni-NTA柱纯化获得纯度大于90%的ScdsFv蛋白。荧光抗体实验和ELISA结果表明ScdsFv具有特异的抗原结合活性,与母本ScFv比较,稳定性有明显提高。这种具有特异抗原结合活性的稳定ScdsFv蛋白的获得为其进一步的功能研究提供了材料。     

单链抗体融合蛋白的构建及应用

通过重组DNA技术将单链抗体(scFv)基因与其他效应蛋白基因融合在一起,经表达后可以得到具有scFv特性和所融合的效应蛋白活性的scFv融合蛋白。这种融合蛋白已应用于许多领域的研究中,并已显示出较高的价值。本就scFv融合蛋白的构建和应用做一综述。     

人源抗狂犬病毒二硫键稳定性Fv抗体的生物学特性研究

对抗狂犬病毒scFv进行稳定性改构 ,纯化制备有活性的人源抗狂犬病毒二硫键稳定性Fv抗体片段 (dsFv) ,然后对其生物学活性进行研究。将dsFvV_H 、V_L 基因在原核表达系统pET22b(+) BL21(DE3)中表达 ,将其包涵体分别在变性缓冲液中溶解 ,稀释后加入折叠缓冲液使其折叠形成有活性的dsFv抗体片段 ,上Ni-NTA柱进行纯化。然后以其亲本scFv作为对照 ,对dsFv的亲和力、稳定性以及体外中和活性进行评价。结果显示 ,与其母本抗体scFv相比 ,改构后的抗狂犬病毒dsFv保持了对狂犬病毒Vero疫苗的特异性识别能力 ,而且其亲和力明显提高 ;抗狂犬病毒dsFv在血清和BSA中的稳定性有明显的改进 ,而且其热稳定性和抵抗尿素化学变性的能力亦大大改进 ;蚀斑减少中和实验显示 ,抗狂犬病毒dsFv抗体片段能特异中和狂犬病毒 ,阻止狂犬病毒对Vero细胞的吸附 ,抑制狂犬病毒对靶细胞的感染 ,从而导致蚀斑减少甚至消失 ;scFv抗体片段仅可部分抑制蚀斑的形成 ,但不能全部抑制。这为进一步研究抗狂犬病毒dsFv基因工程抗体的免疫保护作用及其在临床的应用奠定了基础。     

人源抗狂犬病毒单链抗体基因原核表达质粒的构建及高效表达

在原核系统中高效表达抗狂犬病毒单链抗体scFv41,以便进一步研究其生物学功能,预测临床应用前景。以重组质粒pCANTABscFv41为模板,PCR扩增带NcoI和NotI位点的scFv41基因,克隆入原核表达载体pET-22b( ),酶切鉴定重组表达质粒,转化大肠杆菌BL21(DE3),IPTG诱导表达。竞争ELISA检测表达蛋白的特异结合活性。酶切鉴定证实scFv41基因已插入原核表达载体pETl-22b( ),重组表达质粒pET-scFv41在大肠杆菌BL21(DE3)中获得了高效表达,表达量约占菌体蛋白总量的30％。竞争ELISA检测结果表明scFv41表达蛋白可特异抑制抗狂犬病毒IGY与狂犬病毒的特异性结合。该实验为进一步研究scFv41的生物学特性和免疫保护作用,及基因工程抗体的制备奠定了基础。     

人源抗破伤风毒素单链抗体(ScFv)的原核表达、纯化及功能鉴定

实验通过DNA重组技术从一株可中和破伤风毒素的人源单克隆抗体细胞(G6)中扩增出了抗体VH、VL的基因,通过重叠PCR使连接片段与VH、VL连接成单链ScFv。经测序证实VH、VL为抗体的可变区序列,命名为ScFv-G6。将ScFv-G6连接转化PET/26b质粒,构建了抗体的表达载体,被命名为PET/26b/ScFv-G6。以该载体在大肠杆菌中分泌表达产物经Ni-亲和柱纯化后的小鼠试验证实,可抵抗破伤风毒素的攻击,表明为中和抗体。具有组织穿透力强,不易过敏,可直接靶向于毒素等特点,适合于破伤风的防治,具有重要的应用价值。     

抗人AFP单链抗体与藻胆蛋白融合蛋白的构建、表达与活性分析

目的:构建多基因表达载体,在大肠杆菌中同时表达AFP单链抗体(scFv)和蓝藻别藻蓝蛋白α亚基脱辅基蛋白(apcA)组成的融合蛋白(scFv-apcA)、藻胆蛋白裂合酶(cpcS)及藻红蛋白生物合成酶(Ho1和pebS),获得共价结合藻红胆素的融合蛋白(scFv-apcA-PEB)。方法:利用融合PCR将scFv和apcA基因连接起来,形成scFv-apcA融合基因,并将该融合基因与cpcS克隆到表达载体pCDFDuet-1中;将Ho1和pebS基因克隆到表达载体pRSFDuet-1中。将两种载体共转化到大肠杆菌中,IPTG诱导重组蛋白表达,经亲和层析获得重组蛋白,通过光谱学分析和抗体竞争性抑制法,测定重组蛋白的生物学活性。结果:成功表达融合蛋白scFv-apcA-PEB,分子质量约为45kDa,与理论值相符,其最大吸收峰为549.5nm,最大荧光发射峰为560nm,竞争抑制ELISA法初步鉴定活性,竞争抑制率达到48%。结论:利用大肠杆菌表达系统,获得了同时具有荧光特性和免疫学活性的重组蛋白。     

抗人红细胞单链抗体与猪瘟E2蛋白双功能融合蛋白的构建及生物学活性检测

为构建具有凝集性、免疫反应性的双功能融合蛋白,本研究采用重叠延伸PCR方法将2E8ScFv(抗人红细胞H抗原单链抗体基因)和mE2(猪瘟病毒E2蛋白主要抗原编码区基因)拼接成融合基因2E8mE2,并插入原核表达载体pET-DsbA,将重组表达质粒pET-DsbA-2E8mE2转化入大肠杆菌Escherichia coli BL21(DE3)PlysS中进行IPTG诱导表达,表达的融合蛋白经SDS-PAGE和Western blotting分析鉴定,结果表明:2E8mE2融合基因在大肠杆菌中获得了表达,表达产物以包涵体形式存在,分子量约为65kDa,与预期的大小一致。分别采用亲和层析法和谷胱甘肽再氧化法对融合蛋白进行纯化和复性,红细胞凝集试验证实:2E8mE2融合蛋白复性效果良好,既能够与人红细胞结合,又能够与猪瘟病毒抗体反应,具有双功能特性。     

人源抗狂犬病毒糖蛋白稳定型抗体精氨酸密码子突变株的构建及其在巴斯德毕赤酵母中的分泌表达

目的：对人源抗狂犬病毒糖蛋白（GPRV）单链二硫键稳定抗体（ScdsFv）进行精氨酸密码子修饰,实现其在酵母中的分泌表达,并检测其生物学活性。方法：参照巴斯德毕赤酵母偏好密码子,对抗GPRV ScdsFv原核表达基因进行密码子修饰,并通过点基因融合技术构建ScdsFv重组酵母表达基因,连接pPIC9K构建重组表达质粒pPIC9K-ScdsFv,电转化毕赤酵母GS115,经筛选后进行甲醇诱导表达。结果：SDS-PAGE及Western blot检测到重组表达质粒在30℃经甲醇诱导表达的蛋白相对分子质量为30000;荧光抗体试验验证ScdsFv能靶向结合GPRV;MTT试验说明ScdsFv能中和狂犬病毒,具有一定的细胞保护作用。结论：重组ScdsFv在酵母系统中可以有效表达。具有较好的生物学活性。     

从大容量噬菌体抗体库中筛选抗Acr蛋白人源单链抗体

目的:从天然的大容量噬菌体抗体库中筛选特异的抗结核分枝杆菌晶体蛋白( alpha-crystallin Acr)的人源抗体.方法:以结核分枝杆菌Acr蛋白包被免疫管,通过对噬菌体抗体库进行4轮“吸附-洗脱-扩增”的过程从大容量抗体库中筛选特异性抗结核分枝杆菌Acr蛋白的抗体,并对可变区序列进行了测序分析.将特异性的噬菌体抗体感染HB2151菌,经IPTG诱导表达,制备了抗结核分枝杆菌Acr蛋白的可溶性单链抗体;对其序列和抗原结合活性进行分析鉴定.结果:经过4轮筛选,获得了43个与结核分枝杆菌Acr蛋白结合的阳性克隆,其中29个特异结合的克隆;测序分析有26不同的可变区片段;通过可溶性单链抗体(scFv)表达筛选到14株特异性结合Acr蛋白的可溶性单链抗体克隆;经过基因测序,分析了可变区基因的亚群.成功制备了可溶性单链抗体.Westren blotting分析证实筛选的人源单链抗体能与天然蛋白结合.结论:利用单链大容量抗体库获得抗结核分枝杆菌Acr蛋白的噬菌体抗体并且成功制备抗结核分枝杆菌Acr天然蛋白的可溶性单链抗体,为今后的研究和应用奠定基础.     

抗A型肉毒毒素人源单链抗体融合蛋白的重组设计

以获得的抗A型肉毒毒素单链抗体为模板,进行融合改构,将人IgGl的Fc片段连接到ScFv的C端,在大肠杆菌中实现抗体融合蛋白ScFv—Fc的表达,表达量30％以上,蛋白以包含体形式存在,经过体外变复性的抗体融合蛋白ScFv．Fc,进行Protein G Sepharose柱亲和层析纯化,纯度达90％-95％。体外活性检测结果表明,重组抗体融合蛋白ScFvFc可以特异结合A型肉毒类毒素抗原,其相对亲和力近似于母本单链抗体,其稳定性高于母本单链抗体。     

Structure of trimeric pre-fusion rabies virus glycoprotein in complex with two protective antibodies

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基于对虾白斑综合症病毒极早期启动子的新型杆状病毒表达载体的构建与分析

摘要：【目的】构建携带有受杆状病毒多角体启动子控制的疱疹性口腔炎病毒糖蛋白(vesicular stomatitis virus glycoprotein, VSV G)和受白斑综合症病毒极早期基因(immediately-early gene 1,ie1)启动子控制的绿色荧光蛋白(enhanced green fluorescent protein, EGFP)两个表达阅读框的新型重组病毒vAc-G-EGFP,分析其在无脊椎动物和脊椎动物细胞系中表达报道基因的能力。【方法】 利用Bac-To-Bac 系统构建重组杆状病毒,利用病毒感染或转导实验介导报道基因在待测细胞系中的表达,用荧光显微镜和免疫印迹技术分析报道基因在待测细胞系中的实时表达情况。 【结果】成功构建了分别含VSV G 和 ie1启动子两个阅读框的重组杆状病毒vAc-G-EGFP,发现vAc-G-EGFP可以在无脊椎和脊椎动物细胞系中有效表达报道基因EGFP,免疫印迹实验显示,在不同时间点EGFP于这两类细胞中的表达存在差异。【结论】 基于白斑综合症病毒ie1启动子并携带有VSV G表达框的单一杆状病毒载体可以实现同时在不同种类细胞系中有效表达外源基因。本文构建的新型杆状病毒表达载体有希望普遍应用于基础和应用生物学研究。     

Further studies on the mechanism of rabies virus neutralization by a viral glycoprotein-specific monoclonal antibody, #1-46-12

We previously reported that a conformational epitope-specific monoclonal antibody (mAb; #1-46-12) neutralized the rabies virus by binding only a small number (less than 20) of the antibody molecules per virion, while a linear epitope-specific mAb (#7-1-9) required more than 250 IgG molecules for the neutralization. We also isolated both the epitope-negative (R-31) and-positive (R-61) escape mutants that resisted mAb #1-46-12. Co-infection studies with wild type (wt) and R-61 mutant have shown that although the infectivity of R-61 mutant was not affected by the binding of about 300 IgG molecules per virion, incorporation of a small number of wt G protein into the R-61 virion resulted in dramatic loss of the resistance. In this study, we further investigated properties of the mutant G proteins. The R-61 G protein lost reactivity to the mAb when solubilized, even keeping a trimer form, suggesting that membrane-anchorage is essential for the maintenance of its epitope-positive conformation. On the other hand, incorporation of wt G proteins into the R-31 virions did not affect their resistance to the mAb very much. Although we have not so far found the presumed conformational changes induced by the mAb-binding, we think that these results are not inconsistent with our previously proposed novel model (referred to as a domino effect model) for the virus neutralization by mAb #1-46-12 other than a classical spike-blocking model, which implicates successive spreading of the postulated antibody-induced conformational changes of G protein to the neighboring spikes until abolishing the host cell-binding ability of the virion.     

Characterization of recombinant rabies virus carrying double glycoprotein genes

A recombinant rabies virus carrying double glycoprotein (G) genes, R(NPMGGL) strain, was generated by a reverse genetics system utilizing cloned cDNA of the RC-HL strain, and the biological properties of the virus were compared to those of the recombinant RC-HL (rRC-HL) strain. The extents of virus growth in cultured cells and virulence for adult mice of the R(NPMGGL) strain were almost same as those of the rRC-HL strain, while G protein content of the purified R(NPMGGL) virion and G protein expression level in R(NPMGGL)-infected cells were 1.5-fold higher than those of the rRC-HL strain. As a result of serial passages of the R(NPMGGL) strain in cultured cells, the expression level of G protein in cultured cells infected with the passaged R(NPMGGL) strain was maintained and virus titers rose with adaptation to the cultured cells. Furthermore, analysis of neutralization titers in mice immunized with UVinactivated virus suggested that the R(NPMGGL) strain had higher immunogenicity than that of the rRC-HL strain. The results suggest that the R(NPMGGL) strain carrying double G genes might be a useful candidate for development of a new inactivated rabies vaccine.     

Isolation and characterization of novel human monoclonal antibodies possessing neutralizing ability against rabies virus

Rabies is a fatal viral encephalitis which is transmitted by exposure to the bite of rabid animals. Human and equine rabies immunoglobulins are indispensable pharmacological agents for severe bite exposure, as is vaccine. However, several disadvantages, including limited supply, adverse reactions, and high cost, hamper their wide application in developing countries. In the present study, two novel huMabs which neutralize rabies virus were established from vaccinated hyperimmune volunteers using the Epstein‐Barr virus transformation method. One MAb (No. 254), which was subclass IgG3, effectively neutralized fixed rabies viruses of CVS, ERA, HEP‐Flury, and Nishigahara strains and recognized a well‐conserved epitope located in antigenic site II of the rabies virus glycoprotein. No. 254 possessed 68 ng/ml of FRNT₅₀ activity against CVS, 3.7 × 10^?7 M of the Kd value, and the enhancing effect of complement‐dependent virolysis. In addition, No. 254 showed effective neutralization potency in vivo in the mouse challenge test. The other MAb, 4D4, was subclass IgM and showed neutralizing activity against CVS and Nishigahara strains. 4D4 recognized a novel antigenic site which is associated with the neurovirulence of rabies, a glycoprotein located between antigenic site I and VI. Both human MAbs against rabies are expected to be utilized as a tool for future post‐exposure prophylaxis.     

人源抗狂犬病病毒单克隆抗体的构建及验证

目的利用人源抗狂犬病病毒ScFv噬菌体抗体库,构建单克隆抗体(单抗)轻、重链质粒,瞬时表达,纯化后验证具有高中和活性的单抗。方法以从ScFv噬菌体抗体库中筛选获得特异性抗体为模板,PCR扩增抗体轻、重链可变区序列,构建人源全分子抗体瞬时转染质粒;轻、重链质粒混合后转染HEK293 EBNA1细胞,瞬时表达全分子抗体。采用Mabselect SuRe介质手动亲和纯化抗体,Nano Vue超微量分光光度计测定蛋白质的质量浓度,快速荧光灶抑制试验(rapid fluorescent focus inhibition test, RFFIT)进行体外中和效价验证。结果成功构建22个轻链质粒,15个重链质粒。通过真核细胞瞬时表达后,经手动亲和纯化后,获得22株单抗。除1株抗体外,其余21株抗体的蛋白质量浓度均>300μg/mL;部分抗体纯度均在90%以上。中和活性结果显示,5株在1 500~1 800 IU/mg之间;8株在800~1 500 IU/mg之间;7株在500~800 IU/mg之间;其余2株在500 IU/mg以下。结论成功构建并验证获得20株中和活性>500 IU/mg的人源抗狂犬病病毒单抗,为单抗混合制剂的研究奠定了基础。     

狂犬病病毒Evelyn- Rokitnicki-Abelseth疫苗株反向遗传系统的建立

摘要：【目的】建立狂犬病毒的反向遗传系统,为研制不含狂犬病病毒致病性的新型安全高效的狂犬疫苗提供技术依据。【方法】本研究采用反向遗传学方法和分子克隆技术,建立了狂犬病病毒Evelyn- Rokitnicki-Abelseth (ERA)疫苗株的CMV/ T7、T7启动子病毒拯救系统,构建了表达N、P、L蛋白的辅助质粒。【结果】成功拯救出野生型病毒rERA-VC,在Vero细胞上的生长动力学特性与父母本ERA 相同,第三代可在Vero细胞上可获得很高的生长滴度。【结论】建立了狂犬病病毒的反向遗传系统,拯救出的野生型病毒生物学特性与父母本相同。     

N-glycosylation at one rabies virus glycoprotein sequon influences N-glycan processing at a distant sequon on the same molecule

Rabies glycoprotein (RGP(WT)) contains N-glycosylation sequons at Asn(37), Asn(247), and Asn(319), although Asn(37) is not efficiently glycosylated. To examine N-glycan processing at Asn(247) and Asn(319), full-length glycosylation mutants, RGP(-2-) and RGP(--3), were expressed, and Endo H sensitivity was compared. When the Asn(247) sequon is present alone in RGP(-2-), 90% of its N-glycans are high-mannose type, whereas only 35% of the N-glycans at Asn(319) in RGP(--3) are high-mannose. When both sequons are present in RGP(-23), 87% of the N-glycans are of complex type. The differing patterns of Endo H sensitivity at sequons present individually or together suggests that glycosylation of one sequon affects glycosylation at another, distant sequon. To explore this further, we constructed soluble forms of RGP: RGP(WT)T441His and RGP(--3)T441His. Tryptic glycopeptides from these purified secreted proteins were isolated by HPLC and characterized by a 3D oligosaccharide mapping technique. RGP(WT)T441His had fucosylated, bi- and triantennary complex type glycans at Asn(247) and Asn(319). However, Asn(247) had half as many neutral glycans, more monosialylated glycans, and fewer disialylated glycans when compared with Asn(319). Moreover, when comparing the N-glycans at Asn(319) on RGP(--3)T441His and RGP(WT)T441His, the former had 30% more neutral, 28% more monosialylated, and 33% fewer disialylated glycans. This suggests that the N-glycan at Asn(247) allows additional N-glycan processing to occur at Asn(319), yielding more heavily sialylated bi- and triantennary forms. The mechanism(s) by which glycosylation at one sequon influences N-glycan processing at a distant sequon on the same glycoprotein remains to be determined.     

狂犬病病毒糖蛋白表达及纯化及其记忆性B细胞结合能力的分析

表达纯化不同标签、不同大小3个狂犬病病毒糖蛋白,分析其结合功能后,得到具备高亲和力的、可特异性结合记忆性B细胞的狂犬病病毒糖蛋白。本实验通过基因工程的方法,采用不同的原核表达系统分别表达带有不同标签的、全长和膜外区的RVG,纯化蛋白并分析比较其结合功能,荧光标记候选蛋白,结合CD19及CD27的抗体,流式细胞术检测狂犬疫苗免疫后PBMCs中抗狂犬病病毒特异性记忆性B细胞的情况,确认候选蛋白与抗狂犬病毒特异性记忆性B细胞的结合功能。本实验成功构建了3个表达载体pGEX-5X-1-RVG、pET28a-RVG和pET30a-G,优化表达纯化条件成功获得了糖蛋白GST-RVG、His-RVG和His-G。纯化后的GST-RVG、His-RVG和His-G经Western blotting和ELISA鉴定均有抗原特异性;由竞争ELISA法测得3个纯化后糖蛋白与抗狂犬病病毒抗体的亲和力。通过流式细胞术可以检测到狂犬疫苗免疫后阳性志愿者PBMCs中的抗狂犬病病毒特异性记忆性B细胞,从而获得了高亲和力、可用于分选抗原特异性的记忆性B细胞的狂犬病病毒糖蛋白。