人源抗汉坦病毒核衣壳蛋白单链抗体研究
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摘要
前言
肾综合征出血热(HFRS)是由布尼亚病毒科汉坦病毒属(Hantavirus)的病毒引起的一种高病死率的传染病。我国是受其危害最严重的国家，大约90％的病例发生在我国，由于其临床表现复杂，并发症多，尚无特效治疗方法，其病死率可达5％。目前，汉坦病毒的检测方法还存在许多缺陷，不能为临床治疗提供及时可靠的早期诊断依据，因而，常常错过最佳治疗时机。HFRS致病机理的研究更是进展缓慢，HFRS致病机理还不清楚。因此，研究汉坦病毒的检测和HFRS的治疗与致病机理具有重要的实际意义。
应用噬菌体抗体库技术可筛选出人源抗汉坦病毒抗原抗体，经载体表达出可溶性scFv。可溶性scFv具有穿透力强、容易到达靶细胞等优点，可广泛应用于汉坦病毒的检测及HFRS的治疗和致病机理研究。因此，为表达可溶性抗NP抗原scFv，并初步用于诊断汉坦病毒感染，本研究拟将提取的肾综合征出血热患者外周血淋巴细胞总RNA，扩增V_H和V_L抗体基因，并合成scFv基因，与T7噬菌体载体连接，构建T7噬菌体抗体库，用汉坦病毒76-118核衣壳蛋白(NP)抗原筛选出特异性噬菌体抗体。并从阳性克隆中扩增出scFv基因，克隆入表达载体pGEX-6p-1，表达出可溶性scFv，再初步应用可溶性单链抗体制备双单链抗体夹心试剂，检测血清中汉坦病毒NP抗原，建立敏感特异的肾综合征出血热诊断方法。
实验方法
一、scFv抗体基因的合成
应用淋巴细胞分离液常规分离HFRS恢复期患者外周血淋巴细胞，用RNAout提取总RNA。以总RNA为模板，以oligo(dT)为引物，反转录合成V_H基因和V_L基因的cDNA第一条链。
设计并合成V_H基因和V_L基因两端简并引物，并在V_H基因5’端引物引入EcoRⅠ，在V_L基因引物3’端引入HindⅢ酶切位点。PCR合成V_H基因和V_L基因，以V_H基因和V_L基因为模板，SOE PCR拼接scFv抗体基因。
二、T7噬菌体抗体库的构建
回收SOE PCR扩增scFv基因，用EcoRⅠ和HindⅢ双酶切scFv基因，电泳后凝胶回收双酶切scFv基因与已用EcoRⅠ和HindⅢ双酶切的T7 Select10-3b噬菌体载体的2个臂，经T_4DNA连接酶连接。用T7噬菌体包装提取物包装连接反应产物，铺板测定包装效率和scFv基因插入率。接种宿主菌BLT5403，增殖重组T7噬菌体，铺板测定抗体库的滴度。
三、T7噬菌体抗体库的筛选
以重组汉坦病毒76-118 NP抗原包被酶标板，BSA封闭，加入T7噬菌体抗体库，室温孵育30 min，TBST洗涤5次，1％SDS洗脱抗体库，所得噬菌体抗体即为次级噬菌体抗体库，感染宿主菌BLT5403扩增后，可用于下一轮筛选。滴定次级噬菌体抗体库的滴度，并计算每一轮筛选噬菌体投入／产出比(回收率)作为特异性噬菌体抗体富集的指标，重复上述步骤4次。
四、酶免疫实验检测抗体活性
将上述筛选所得的抗体库铺板，随机选取噬菌斑，在BLT5403内扩增，以NaCl和PEG 8000纯化噬菌体抗体。用1％戊二醛偶联噬菌体抗体和辣根过氧化物酶(HRP)，加过量10％甘氨酸封闭未反应的戊二醛。同时，将野生噬菌体与辣根过氧化物酶偶联，作为阴性对照。将酶偶联噬菌体抗体和阴性对照，分别加入汉坦病毒G_1、G_2包膜蛋白和NP抗原包被的酶标板，37℃孵育1h，TBST洗涤5次，加邻苯二胺和H_2O_2底物，37℃作用15min，2M H_2SO_4终止反应，在492nm波长用酶标仪检测OD值，样品A_(492)／阴性对照A_(492)＞2者，为阳性。
五、pGEX-6P-1-scFv重组质粒的构建
选择抗体活性最高T7噬菌体抗体在BLT5403中增殖，酚氯仿法提取T7 DNA，对T7噬菌体中scFv进行测序。设计并合成scFv基因简并引物，并在其5’端和3’端引物中引入BamHⅠ和SalⅠ切酶位点。以提取的T7噬菌体DNA为模板，以合成的scFv简并引物为引物，PCR扩增scFv基因。回收PCR扩增的scFv基因，用BamHⅠ和SalⅠ双酶切scFv基因和pGEX-6p-1载体，电泳后凝胶回收双酶切scFv基因和pGEX-6p-1载体大片段，16℃连接4小时，转化E．coli BL-21(DE3)感受态菌。涂于Amp平板培养基，37℃培养过夜。
六、重组质粒的筛选及鉴定
挑取单个菌落，Amp LB液体培养基培养，碱裂解法粗提质粒，0.7％琼脂糖凝胶电泳初筛阳性质粒，再用BamHⅠ和SalⅠ分别双酶切及PCR扩增法对重组质粒进行鉴定并对scFv测序。
七、scFv融合蛋白的表达和纯化及SDS-PAGE检测和酶免疫检测
LB(含50μg／ml Amp)液体培养基培养含有pGEX-6P-1-scFv质粒的E．coliBL-21(DE3)菌株。加入IPTG至终浓度为1mM，诱导scFv融合蛋白表达。离心收集菌体，悬浮于PBS，超声破碎。GST凝胶亲和层析纯化scFv融合蛋白。常规SDS-PAGE检测scFv融合蛋白。
酶免疫鉴定可溶性单链抗体scFv生物活性，方法同四、。
八、酶标抗体的制备和血清标本检测
1％戊二醛偶联辣根过氧化物酶和纯化的可溶性单链抗体，并用聚乙二醇浓缩。以可溶性抗NP单链抗体包被酶标板，BSA封闭酶标板。样品血清每孔加样50μl，37℃反应1h，PBST洗涤5次，加酶标抗体100μl，PBST洗涤5次。显色及结果判断同步骤四、。
结果
一、scFv抗体基因的合成
用淋巴细胞分离液共分离得到1.5×10~7肾综合征出血热恢复期患者外周血淋巴细胞。RNAout提取总RNA，电泳后呈明显的3条带，即28 S RNA，18 S RNA和5 S RNA，A_(260)nm／A_(280)nm的比值为1.8，说明提取的总RNA完整且纯度好。RT-PCR扩增V_H基因和V_L基因，两者分子大小约为400bp，与理论估计值大小一致，用SOE PCR将V_H基因和V_L基因连接成scFv基因，分子大小约为750bp，与理论估计值大小一致。
二、T7噬菌体抗体库的构建
噬菌体抗体库构建后，测定DNA包装效率为1.5×10~7／μg DNA。测定scFv基因的插入率为90％，其库容为1.35×10~7。初级噬菌体抗体库滴度为2.12×10~(10)PFU／mL。
三、T7噬菌体抗体库的筛选
用NP抗原对噬菌体抗库进行4轮“吸附-洗脱-扩增”的筛选，噬菌体抗体得到约60倍的富集。
四、酶免疫实验检测抗体活性
随机挑取第4轮筛选产物23个克隆，与辣根过氧化物酶偶联，与用汉坦病毒G_1、G_2包膜蛋白和NP抗原包被的酶标板特异性反应。酶免疫结果显示，19株具有抗原特异性结合活性，其中有2株具有较高的NP抗原特异性结合活性。
五、pGEX-6P-1-scFv重组质粒的构建
将亲和力最高的T7噬菌体抗体，在BLT5403中增殖。提取T7噬菌体DNA，干燥后溶于TE，1％琼脂糖凝胶电泳检测，可见36 kbT7 DNA噬菌体。测序证实获得scFv序列。以T7噬菌体DNA为模板，PCR扩增scFv基因，经0.7％琼脂糖凝胶电泳检测为750bp，与预期的scFv片段大小相同。
六、重组质粒的筛选及鉴定
碱裂解法粗提质粒，0.7％琼脂糖凝胶电泳初筛阳性质粒，阳性质粒经BamHⅠ和SalⅠ双酶切后，成5.0kb和750bp两条明显条带，酶切鉴定结果表明成功构建了pGEX-6P-1-scFv重组质粒。同时通过PCR扩增方法对重组质粒进行了鉴定，获得了750bp扩增产物，证实获得了含scFv基因片段的重组质粒。测序结果显示，质粒中scFv与T7噬菌体相同。
七、scFv融合蛋白的表达和纯化及SDS-PAGE检测和酶免疫检测
将含有pGEX-6P-1-scFv质粒的E．coliBL-21(DE3)菌株培养，加入IPTG诱导，诱导融合蛋白表达，超声破碎菌体，GST凝胶于层析柱纯化单链抗体融合蛋白。10％SDS-PAGE凝胶电泳，检测到纯化的融合scFv分子量大小为56KDa，与预期的结果相同，酶免疫检测具有NP抗原结合功能。
八、酶标抗体的制备和血清标本检测
以1％戊二醛溶液偶联纯化的可溶性scFv和HRP，聚乙二醇浓缩酶标记抗体至1 ml。双单链抗体夹心法检测56份HFRS患者血清中，检测出29例阳性，阳性率为51.79％，对照组66例，检测结果均为阴性。
讨论
本研究所构建噬菌体抗体库库容为1.35×10~7，scFv基因的插入率为90％，测定DNA包装效率为1.5×10~7／μg DNA，初级噬菌体抗体库的滴度为2.12×10~(10)PFU／mL，经过4轮“吸附-洗脱-扩增”的筛选噬菌体得到约60倍的富集，酶免疫实验显示有2株具有较高的NP抗原特异性结合活性。这些数据与国内姚龙泉等学者所建文库非常相似。
T7噬菌体优点是所展示的蛋白质的分子量很大，其最大展示蛋白的分子为1200氨基酸，这是M13噬菌体无法达到的。T7噬菌体的另一大优点是它极为稳定，能在各种严酷的条件下不被破坏，而其它噬菌体常在亲和淘洗过程中失活。此外，T7生长极为迅速，T7噬菌体形成噬斑仅需3小时，这可以大大缩短研究周期。
在引物设计方面，设计一对V_H简并引物扩增出大部分V_H基因，设计一对V_L简并引物扩增出大部分V_L基因，既最大限度地扩增出了多样的抗体基因，又减少了引物的使用，简化了操作，节约了时间。同时在V_H的羧基端基因和V_L的氨基端基因引入编码连接肽(Gly_4Ser)_3序列，通过SOE PCR法直接将V_H基因和V_L基因拼接成V_H-(Gly_4Ser)_3-V_L形式的scF_v，这样大大简化了基因重组过程。
目前大多数研究只是从噬菌体库中筛选出相映抗体。而本研究进一步将scFv基因克隆到pGEX-6P-1上，表达出可以最终应用的可溶性单链抗体。融合scFv分子量约为56Kda，包括30Kda单链抗体和26Kda载体蛋白，载体蛋白一般不影响单链抗体的功能，而且载体蛋白有利于蛋白纯化，必要时可以被蛋白酶完全切掉。
双单链抗体夹心法，与传统的双抗体夹心法相比其最大优势之一是其造价低廉。通过基因工程技术，在大肠杆菌中生产出单链抗体比通过杂交瘤技术在培养细胞中生产的单克隆抗体更节约时间且成本低廉，还容易操作。
双单链抗体夹心法检测56份患者血清和对66例对照，在56份HFRS患者血清中，检测出29例阳性，阳性率为51.79％，在对照组中为检测结果均为阴性。目前认为，病程早期以病毒血症为主，在肾综合征出血热的病程晚期，机体免疫功能发挥作用，病毒逐渐被清除，不易在患者血清中检出病毒抗原。由此推测，本研究中25例未检出NP抗原标本可能在病程晚期。
结论
本研究运用噬菌体抗体库技术构建了人源单链T7噬菌体抗体库，库容量为1.35×10~7，初级噬菌体抗体库的滴度为2.12×10~(10)PFU／mL。经酶免疫实验筛选到2株具有较高的与NP抗原特异性结合活性噬菌体抗体。研究中成功构建了融合蛋白表达载体pOEX-6P-1-scFv，获得了高效表达的可溶性scFv，酶免疫实验显示，可溶性scFv具有较高的NP抗原特异性结合活性。首次用大肠杆菌表达的抗NP单链抗体，成功制备了双单链抗体夹心诊断试剂，成功用于诊断汉坦病毒感染。
Introduction
Hemorrhagic fever with renal syndrome (HFRS) is an infectious disease with high mortality rate caused by virus of the genus Hantavirus in the family Bunyavirdae. Our country suffers from it most seriously in the world. About 90 percent of HFRS case happened in our country. Due to its complex symptom, various complications and having no specific therapies, the mortality rate of HFRS is about 5 percent. Now the detecting method of Hantavirus had some blemish so they can't offer reliable diagnosis of early infection for therapy, therefore best opportunity of therapy often missed. The research on pathogenesis of HFRS made less progress, and pathogenesis of HFRS was not illustrated yet. Therefore, the researches on diagnosis of Hantavirus and therapy and pathogenesis of HFRS are very important.
The human phage antibody against antigen of Hantavirus can be screened out by phage display technique and then soluble scFv can be expressed in vector. Due to advantages of strong penetration and easy reaching target cell, soluble scFv is enabled extensive potent application in the research on diagnosis of Hantavirus and therapy and pathogenesis of HFRS. Therefore, in order to express soluble anti-NP scFv antibody and apply it in diagnosis of Hantavirus infection primarily, V_H gene and V_L gene were amplified by RT-PCR with template of total RNA extracted from peripheral lymphocyte of HFRS patients during the recovery stage. The scFv gene was constructed by SOE PCR from Vngene and V_L gene, and then ligated into T7 phage vector. The specific phage antibody was panned with expressed 76-118 nucleocapsid protein (NP)of hantaan. After amplified from positive clone, the scFv gene was cloned into pGEX-6p-l vector to express soluble scFv. Double scFv sandwich ELISA reagent was prepared with soluble scFv to detect NP antigen in sera as primary application of soluble scFv to establish a sensitive and specific diagnosis method for HFRS.
Method
一、Synthesis of scFv gene
The peripheral lymphocyte of HFRS patients during the recovery stage was separated by lymphocyte separation medium. Total RNA was extracted with RNAout. The first strands of cDNA of V_H gene and V_L gene were synthesized by reverse transcription assay with primer of oligo (dT) and template of total RNA.
The degenerate primers of V_H gene and V_L gene were designed and synthesized with EcoR I site being added into 5' terminal primer of V_H gene and HindIII site being added into the antisense primer of V_L gene. After V_H gene and V_L gene were amplified by PCR, scFv gene was constructed by SOE PCR with template of V_H gene and V_L gene.
二、Construction of T7 phage library
After being purified , scFv gene was digested with EcoR I and Hind III, and then analyzed by electrophoresis and purified again. The digested scFv gene was ligated with 2 arms of T7 Select10-3b digested with EcoR I and Hind III by T_4DNA ligase. The ligation product was packaged with T7 packaging extract. Packaging efficiency and scFv gene inserting rate were detected after plaque assay. The recombinant T7 phage was propagated in host BLT5403. The titer of the phage library was detected by plaque assay.
三、Panning of T 7 phage library
The ELISA plate was coated with recombinant NP antigen of Hantavirus 76-118 and blocked with BSA. After applied with T7 phage library and incubated for 30 min at room temperature, the well was washed 5 times with TBST and then bound phage was eluted with 1%SDS. The eluted phage was next round phage library that can be applied for next round screening after propagation in host BLT5403. The titter of next round phage library was calculated and the multiplicity of each round was calculated according to input and output of library. Above operation was repeated 4 times.
四、Identification of individual clones by enzyme immunoassay
After panning, the library was detected by plaque assay. After plaques were scraped at random, individual clones were propagated in BLT5403, and then purified with NaCl and PEG 8000. Phage antibody and peroxidase horseradish (HRP) were conjugated by 1% glutaraldehyde and then 10% superfluous glycine was added to block superfluous glutaraldehyde. At same time, wild type phage and peroxidase horseradish (HRP) were conjugated to serve as negative control. The HRP-conjugated phage antibody and negative control were applied to ELISA plate coated with NP antigens G1 glycoprotein and G2 glycoprotein of Hantavirus. After incubated for 1 hour at 37℃, the wells were washed 5 times with TBST. The substrates of o-Diaminobenzene and H_2O_2 were applied into each well, and after incubating for 15min at 37℃, 2M H_2SO_4 was added to terminate reaction. OD of each well was detected at 492nm wave length. If specimen A_(492)/ negative control A_(492) was above 2, the specimen was positive.
五、Construction of recombinant pGEX-6P-1- scFv
The phage antibody with highest activity was propagated in BLT5403, and T7 DNA was extracted with phenol-chloroform, and then scFv gene in T7 DNA was sequenced. The degenerate primers of scFv gene were designed and synthesized with BamH I site being added into 5' terminal primer and Sal I site being added into 3' terminal primer. The scFv gene was amplified by PCR with a template of T7 DNA and two primers of synthesized degenerate primers. The scFv gene of PCR product was purified. scFv gene and pGEX-6p-1 vector were digested with BamH I and Sal I , and analyzed by electrophoresis and purified. The digested scFv gene and large fragment of pGEX-6p-1 were ligated by T_4DNA ligase at 16℃for 4 h. The recombinant plasmid was transformed into competent E.coliBL-21 (DE3) and then cultured in Amp plate medium at 37℃overnight.
六、Screening and identification of recombinant plasmid
After being scraped, the individual clones were cultivated in Amp LB liquid medium. Recombinant plasmid was extracted by alkali lysis, and then identified by 0.7% agarose gel electrophoresis to find out candidate of positive clone. The candidate of positive clone was identified by digestion of BamH I and Sal I and PCR amplification. scFv gene in recombinant plasmid was sequenced.
七、Expression and purification of fusion scFv and identificationof scFv by SDS-PAGE and enzyme immunoassay
E.coliBL-21(DE3) ,which contained the pGEX-6P-1-NP-scFv, was cultivated in LB liquid medium supplemented with 50μg of ampicillin per ml. Culture was induced to produce fusion scFv by addition of IPTG to 1mM. After inducement, the culture was centrifuged to collect bacterial cells. The bacterial cells were re-suspended in PBS and lysed by ultrasonication. The fusion scFv was purified by a GST affinity chromatography column and was identified by SDS-PAGE.The soluble scFv was identified by enzyme immunoassay according to step四、.
八、Preparation of HRP-labeled scFv and Detection of sera specimen
The purified soluble scFv and HRP were conjugated by 1% glutaraldehyde and then condensed by PEG. ELISA plate was coated with soluble scFv and was blocked with BSA. Each well was applied with 50μl serum specimen. After incubated for 1 h at 37℃and washed 5 time with PBST , each well was applied with 100μl HRP-labeled scFv, and the well was washed 5 times with TBST. Following operation was as same as step四.
Result
一、Synthesis of scFv gene
1.5×10~7 peripheral lymphocyte of HFRS patients during the recovery stage was separated by lymphocyte separation medium. After being extracted with RNAout, the total RNA was identified by electrophoresis and 3 bands of 28 S RNA 18S RNA and 5 S RNA were obvious, and A_(260)nm/ A_(280)nm ratio was 1.8. These data indicated that integrity and purity of total RNA was very fine. After synthesized by RT- PCR , V_H gene and V_L gene were identified by electrophoresis, and the molecular weight (MW) of V_H gene and V_L gene was about 400 bp similar to the data in theory. After synthesized by SOE PCR by splicing V_H gene and V_L together, scFv gene was identified by electrophoresis and MW of scFv gene was about 750 bp similar to the data in theory.
二、Construction of T7 phage library
After construction of T7 phage library, efficiency of package was 1.5×10~7/μg DNA, and inserting rate of scFv gene was 90%, and the size of the library was 1.35×10~7, and titer of the primary phage library was 2.12×10~(10)PFU/mL.
三、Panning of T 7 phage library
The library was panned by 4 rounds of " absorption-elution-propagation" with NP antigen and the phage was enriched by 60 times.
四、Identification of individual clones by enzyme immunoassay
After the forth round screening, 23 individual clones were conjugated with HRP, and applied into ELISA plate coated with NP G1 glycoprotein and G2 glycoprotein of Hantavirus. Enzyme immunoassay shows that 19 clones with ability of specific binding to NP were identified and 2 of them had high ability of specific binding to NP antigen.
五、Construction of recombinant pGEX-6P-1- scFv
The phage antibody with highest activity was propagated in BLT5403. T7 phage DNA was extracted and after drying in air dissolved in TE buffer. T7 DNA was identified by 1% agarose gel electrophoresis and 36 kb T7 DNA was found. Sequencing demonstrated that scFv was gained. scFv gene was amplified by PCR with a template of T7 DNA, and scFv gene was identified by 0.7% agarose gel electrophoresis and 750bp band was found similar to the data in expectation.
六、Screening and identification of recombinant plasmid
Recombinant plasmid extracted by alkali lysis was identified by 0.7% agarose gel electrophoresis to find out candidate of positive clone. The candidate of positive clone was digested with BamH I and Sal I and identified by 0.7% agarose gel electrophoresis and 2 band of 5.0kb and 750bp was found. The result indicated that recombinant pGEX-6P-1- scFv was constructed successfully. At same time, scFv gene was amplified by PCR, and identified by 1% agarose gel electrophoresis and 750bp band was found. The data show that recombinant plasmid, which contained scFv, was constructed successfully. Sequencing showed scFv in recombinant plasmid was as same as scFv in T7 DNA.
七、Expression and purification of fusion scFv and identificationof scFv by SDS-PAGE and enzyme immunoassay
E.coliBL-21(DE3) ,which contained the pGEX-6P-1-NP-scFv, was cultivated in LB and induced to produce fusion scFv by addition of IPTG. The bacterial cells were lysed by ultrasonication and the fusion scFv was purified by a GST affinity chromatography column. The purified fusion scFv was identified by 10% SDS-PAGE electrophoresis and result show a 56Kda fusion scFv was found similar to expectation. Enzyme immunoassay shows that fusion scFv had ability of specific binding to NP antigen.
八、Preparation of HRP-labeled scFv and Detection of seraspecimen
The purified soluble scFv and HRP were conjugated by 1% glutaraldehyde and HRP-labeled scFv was condensed to 1ml by PEG. 56 specimens of sera, of which 29 sera were positive, of HFRS patients were detected by double scFv sandwich and positive rate was 51.79%. 66 specimens of sera in the control group were all negative.
Discussion
A phage display library was constructed with size of 1 .35×10~7, and inserting rate of scFv gene was 90%, and efficiency of package was 1.5×10~7/μg DNA, The titer of the primary phage library was 2.12×10~(10)PFU/mL. After 4 rounds of "absorption-elution-propagation" , the phage was enriched by 60 times. 2 clones with high ability of specific binding to NP antigen were identified by enzyme immunoassay. All the data were similar to the data in other researches conducted by Yao Longquan and others.
One of T7 phage's advantage is that it can display a very big exogenous protein and the biggest exogenous protein displayed by T7 phage is about 1200 amino acid while M13 phage can't. Other advantage of T7 phage is that it is so stable that it can't be ruined in severe condition, on the contrary, many other kinds of phages were inactivated during panning. Finally, T7 phage grows so quickly that plaque can be formed in only 3 hours, and this leads to reducing research time terrifically.
A pair of degenerate primers of V_H gene were designed to amplify most V_H gene and a pair of degenerate primers of V_L gene were designed to amplify most V_L gene. Not only antibody genes were amplified as more various as possible but also less primer was used, so that operation was simplified and time was saved. At same time, a sequence of gene of linkage peptide (Gly_4Ser)_3 was added into carboxylic terminal of V_H gene and amino terminal of V_L gene. V_H gene and V_L gene was joined directly to be scFv by SOE PCR as V_H -(Gly_4Ser)_3- V_L so the manipulation of gene was simplified greatly.
Now most research on phage display library ended after gaining corresponding antibody by panning. In this research, scFv gene was cloned into pGEX-6P-1 and soluble fusion scFv, which can be applied finally in following research, was expressed. Molecular weight of fusion scFv was about 56Kda,including scFv of 30Kda and vector protein of 26Kda. The vector protein normally did not affect the function of scFv, on the contrary, it is useful for purification of fusion scFv and it can be cut off by protease if necessary.
One of its biggest advantages of double scFv sandwich lied in its less cost compared with traditional double antibody sandwich. Compared with production of monoclonal antibody in cell by hybridoma technique, production of scFv in E. coli by gene-engineering can save more time and money, and is easier to operate.
56 sera specimen of HFRS patients and 66 sera in control groups were detected by double scFv sandwich. 29 sera from 56 sera specimen of HFRS patients were positive and positive rate was 51.79%, and all sera in control groups were negative. It is believed that patients are in viremia during early stage of HFRS and virus is gradually removed by immune system during the late stage of HFRS, so it is not easy to detected virus antigen in sera during the late stage of HFRS. So it was assumed that the 25 sera, which were negative in detection, of HFRS patients belong to patients during late stage of HFRS.
Conclusion
In this research, a T7 phage display library of human scFv was constructed with the size of 1.35×10~7, and the titer of the primary library was 2.12×10~(10)PFU/mL. 2 clones with high ability of specific binding to NP antigen were identified by enzyme immunoassay. In this research, recombinant vector pGEX-6P-1- scFv, which can express fusion protein, was constructed successfully. Soluble scFv was expressed efficiently and enzyme immunoassay showed that the soluble scFv has high ability of specific binding to NP antigen. In this research, the double scFv sandwich ELISA reagent was prepared first time with anti-NP scFv expressed in E. coli, and was applied to diagnose Hantavirus infection successfully.
肾综合征出血热(HFRS)是由布尼亚病毒科汉坦病毒属(Hantavirus)的病毒引起的一种高病死率的传染病。我国是受其危害最严重的国家，大约90％的病例发生在我国，由于其临床表现复杂，并发症多，尚无特效治疗方法，其病死率可达5％。目前，汉坦病毒的检测方法还存在许多缺陷，不能为临床治疗提供及时可靠的早期诊断依据，因而，常常错过最佳治疗时机。HFRS致病机理的研究更是进展缓慢，HFRS致病机理还不清楚。因此，研究汉坦病毒的检测和HFRS的治疗与致病机理具有重要的实际意义。
应用噬菌体抗体库技术可筛选出人源抗汉坦病毒抗原抗体，经载体表达出可溶性scFv。可溶性scFv具有穿透力强、容易到达靶细胞等优点，可广泛应用于汉坦病毒的检测及HFRS的治疗和致病机理研究。因此，为表达可溶性抗NP抗原scFv，并初步用于诊断汉坦病毒感染，本研究拟将提取的肾综合征出血热患者外周血淋巴细胞总RNA，扩增V_H和V_L抗体基因，并合成scFv基因，与T7噬菌体载体连接，构建T7噬菌体抗体库，用汉坦病毒76-118核衣壳蛋白(NP)抗原筛选出特异性噬菌体抗体。并从阳性克隆中扩增出scFv基因，克隆入表达载体pGEX-6p-1，表达出可溶性scFv，再初步应用可溶性单链抗体制备双单链抗体夹心试剂，检测血清中汉坦病毒NP抗原，建立敏感特异的肾综合征出血热诊断方法。
实验方法
一、scFv抗体基因的合成
应用淋巴细胞分离液常规分离HFRS恢复期患者外周血淋巴细胞，用RNAout提取总RNA。以总RNA为模板，以oligo(dT)为引物，反转录合成V_H基因和V_L基因的cDNA第一条链。
设计并合成V_H基因和V_L基因两端简并引物，并在V_H基因5’端引物引入EcoRⅠ，在V_L基因引物3’端引入HindⅢ酶切位点。PCR合成V_H基因和V_L基因，以V_H基因和V_L基因为模板，SOE PCR拼接scFv抗体基因。
二、T7噬菌体抗体库的构建
回收SOE PCR扩增scFv基因，用EcoRⅠ和HindⅢ双酶切scFv基因，电泳后凝胶回收双酶切scFv基因与已用EcoRⅠ和HindⅢ双酶切的T7 Select10-3b噬菌体载体的2个臂，经T_4DNA连接酶连接。用T7噬菌体包装提取物包装连接反应产物，铺板测定包装效率和scFv基因插入率。接种宿主菌BLT5403，增殖重组T7噬菌体，铺板测定抗体库的滴度。
三、T7噬菌体抗体库的筛选
以重组汉坦病毒76-118 NP抗原包被酶标板，BSA封闭，加入T7噬菌体抗体库，室温孵育30 min，TBST洗涤5次，1％SDS洗脱抗体库，所得噬菌体抗体即为次级噬菌体抗体库，感染宿主菌BLT5403扩增后，可用于下一轮筛选。滴定次级噬菌体抗体库的滴度，并计算每一轮筛选噬菌体投入／产出比(回收率)作为特异性噬菌体抗体富集的指标，重复上述步骤4次。
四、酶免疫实验检测抗体活性
将上述筛选所得的抗体库铺板，随机选取噬菌斑，在BLT5403内扩增，以NaCl和PEG 8000纯化噬菌体抗体。用1％戊二醛偶联噬菌体抗体和辣根过氧化物酶(HRP)，加过量10％甘氨酸封闭未反应的戊二醛。同时，将野生噬菌体与辣根过氧化物酶偶联，作为阴性对照。将酶偶联噬菌体抗体和阴性对照，分别加入汉坦病毒G_1、G_2包膜蛋白和NP抗原包被的酶标板，37℃孵育1h，TBST洗涤5次，加邻苯二胺和H_2O_2底物，37℃作用15min，2M H_2SO_4终止反应，在492nm波长用酶标仪检测OD值，样品A_(492)／阴性对照A_(492)＞2者，为阳性。
五、pGEX-6P-1-scFv重组质粒的构建
选择抗体活性最高T7噬菌体抗体在BLT5403中增殖，酚氯仿法提取T7 DNA，对T7噬菌体中scFv进行测序。设计并合成scFv基因简并引物，并在其5’端和3’端引物中引入BamHⅠ和SalⅠ切酶位点。以提取的T7噬菌体DNA为模板，以合成的scFv简并引物为引物，PCR扩增scFv基因。回收PCR扩增的scFv基因，用BamHⅠ和SalⅠ双酶切scFv基因和pGEX-6p-1载体，电泳后凝胶回收双酶切scFv基因和pGEX-6p-1载体大片段，16℃连接4小时，转化E．coli BL-21(DE3)感受态菌。涂于Amp平板培养基，37℃培养过夜。
六、重组质粒的筛选及鉴定
挑取单个菌落，Amp LB液体培养基培养，碱裂解法粗提质粒，0.7％琼脂糖凝胶电泳初筛阳性质粒，再用BamHⅠ和SalⅠ分别双酶切及PCR扩增法对重组质粒进行鉴定并对scFv测序。
七、scFv融合蛋白的表达和纯化及SDS-PAGE检测和酶免疫检测
LB(含50μg／ml Amp)液体培养基培养含有pGEX-6P-1-scFv质粒的E．coliBL-21(DE3)菌株。加入IPTG至终浓度为1mM，诱导scFv融合蛋白表达。离心收集菌体，悬浮于PBS，超声破碎。GST凝胶亲和层析纯化scFv融合蛋白。常规SDS-PAGE检测scFv融合蛋白。
酶免疫鉴定可溶性单链抗体scFv生物活性，方法同四、。
八、酶标抗体的制备和血清标本检测
1％戊二醛偶联辣根过氧化物酶和纯化的可溶性单链抗体，并用聚乙二醇浓缩。以可溶性抗NP单链抗体包被酶标板，BSA封闭酶标板。样品血清每孔加样50μl，37℃反应1h，PBST洗涤5次，加酶标抗体100μl，PBST洗涤5次。显色及结果判断同步骤四、。
结果
一、scFv抗体基因的合成
用淋巴细胞分离液共分离得到1.5×10~7肾综合征出血热恢复期患者外周血淋巴细胞。RNAout提取总RNA，电泳后呈明显的3条带，即28 S RNA，18 S RNA和5 S RNA，A_(260)nm／A_(280)nm的比值为1.8，说明提取的总RNA完整且纯度好。RT-PCR扩增V_H基因和V_L基因，两者分子大小约为400bp，与理论估计值大小一致，用SOE PCR将V_H基因和V_L基因连接成scFv基因，分子大小约为750bp，与理论估计值大小一致。
二、T7噬菌体抗体库的构建
噬菌体抗体库构建后，测定DNA包装效率为1.5×10~7／μg DNA。测定scFv基因的插入率为90％，其库容为1.35×10~7。初级噬菌体抗体库滴度为2.12×10~(10)PFU／mL。
三、T7噬菌体抗体库的筛选
用NP抗原对噬菌体抗库进行4轮“吸附-洗脱-扩增”的筛选，噬菌体抗体得到约60倍的富集。
四、酶免疫实验检测抗体活性
随机挑取第4轮筛选产物23个克隆，与辣根过氧化物酶偶联，与用汉坦病毒G_1、G_2包膜蛋白和NP抗原包被的酶标板特异性反应。酶免疫结果显示，19株具有抗原特异性结合活性，其中有2株具有较高的NP抗原特异性结合活性。
五、pGEX-6P-1-scFv重组质粒的构建
将亲和力最高的T7噬菌体抗体，在BLT5403中增殖。提取T7噬菌体DNA，干燥后溶于TE，1％琼脂糖凝胶电泳检测，可见36 kbT7 DNA噬菌体。测序证实获得scFv序列。以T7噬菌体DNA为模板，PCR扩增scFv基因，经0.7％琼脂糖凝胶电泳检测为750bp，与预期的scFv片段大小相同。
六、重组质粒的筛选及鉴定
碱裂解法粗提质粒，0.7％琼脂糖凝胶电泳初筛阳性质粒，阳性质粒经BamHⅠ和SalⅠ双酶切后，成5.0kb和750bp两条明显条带，酶切鉴定结果表明成功构建了pGEX-6P-1-scFv重组质粒。同时通过PCR扩增方法对重组质粒进行了鉴定，获得了750bp扩增产物，证实获得了含scFv基因片段的重组质粒。测序结果显示，质粒中scFv与T7噬菌体相同。
七、scFv融合蛋白的表达和纯化及SDS-PAGE检测和酶免疫检测
将含有pGEX-6P-1-scFv质粒的E．coliBL-21(DE3)菌株培养，加入IPTG诱导，诱导融合蛋白表达，超声破碎菌体，GST凝胶于层析柱纯化单链抗体融合蛋白。10％SDS-PAGE凝胶电泳，检测到纯化的融合scFv分子量大小为56KDa，与预期的结果相同，酶免疫检测具有NP抗原结合功能。
八、酶标抗体的制备和血清标本检测
以1％戊二醛溶液偶联纯化的可溶性scFv和HRP，聚乙二醇浓缩酶标记抗体至1 ml。双单链抗体夹心法检测56份HFRS患者血清中，检测出29例阳性，阳性率为51.79％，对照组66例，检测结果均为阴性。
讨论
本研究所构建噬菌体抗体库库容为1.35×10~7，scFv基因的插入率为90％，测定DNA包装效率为1.5×10~7／μg DNA，初级噬菌体抗体库的滴度为2.12×10~(10)PFU／mL，经过4轮“吸附-洗脱-扩增”的筛选噬菌体得到约60倍的富集，酶免疫实验显示有2株具有较高的NP抗原特异性结合活性。这些数据与国内姚龙泉等学者所建文库非常相似。
T7噬菌体优点是所展示的蛋白质的分子量很大，其最大展示蛋白的分子为1200氨基酸，这是M13噬菌体无法达到的。T7噬菌体的另一大优点是它极为稳定，能在各种严酷的条件下不被破坏，而其它噬菌体常在亲和淘洗过程中失活。此外，T7生长极为迅速，T7噬菌体形成噬斑仅需3小时，这可以大大缩短研究周期。
在引物设计方面，设计一对V_H简并引物扩增出大部分V_H基因，设计一对V_L简并引物扩增出大部分V_L基因，既最大限度地扩增出了多样的抗体基因，又减少了引物的使用，简化了操作，节约了时间。同时在V_H的羧基端基因和V_L的氨基端基因引入编码连接肽(Gly_4Ser)_3序列，通过SOE PCR法直接将V_H基因和V_L基因拼接成V_H-(Gly_4Ser)_3-V_L形式的scF_v，这样大大简化了基因重组过程。
目前大多数研究只是从噬菌体库中筛选出相映抗体。而本研究进一步将scFv基因克隆到pGEX-6P-1上，表达出可以最终应用的可溶性单链抗体。融合scFv分子量约为56Kda，包括30Kda单链抗体和26Kda载体蛋白，载体蛋白一般不影响单链抗体的功能，而且载体蛋白有利于蛋白纯化，必要时可以被蛋白酶完全切掉。
双单链抗体夹心法，与传统的双抗体夹心法相比其最大优势之一是其造价低廉。通过基因工程技术，在大肠杆菌中生产出单链抗体比通过杂交瘤技术在培养细胞中生产的单克隆抗体更节约时间且成本低廉，还容易操作。
双单链抗体夹心法检测56份患者血清和对66例对照，在56份HFRS患者血清中，检测出29例阳性，阳性率为51.79％，在对照组中为检测结果均为阴性。目前认为，病程早期以病毒血症为主，在肾综合征出血热的病程晚期，机体免疫功能发挥作用，病毒逐渐被清除，不易在患者血清中检出病毒抗原。由此推测，本研究中25例未检出NP抗原标本可能在病程晚期。
结论
本研究运用噬菌体抗体库技术构建了人源单链T7噬菌体抗体库，库容量为1.35×10~7，初级噬菌体抗体库的滴度为2.12×10~(10)PFU／mL。经酶免疫实验筛选到2株具有较高的与NP抗原特异性结合活性噬菌体抗体。研究中成功构建了融合蛋白表达载体pOEX-6P-1-scFv，获得了高效表达的可溶性scFv，酶免疫实验显示，可溶性scFv具有较高的NP抗原特异性结合活性。首次用大肠杆菌表达的抗NP单链抗体，成功制备了双单链抗体夹心诊断试剂，成功用于诊断汉坦病毒感染。
Introduction
Hemorrhagic fever with renal syndrome (HFRS) is an infectious disease with high mortality rate caused by virus of the genus Hantavirus in the family Bunyavirdae. Our country suffers from it most seriously in the world. About 90 percent of HFRS case happened in our country. Due to its complex symptom, various complications and having no specific therapies, the mortality rate of HFRS is about 5 percent. Now the detecting method of Hantavirus had some blemish so they can't offer reliable diagnosis of early infection for therapy, therefore best opportunity of therapy often missed. The research on pathogenesis of HFRS made less progress, and pathogenesis of HFRS was not illustrated yet. Therefore, the researches on diagnosis of Hantavirus and therapy and pathogenesis of HFRS are very important.
The human phage antibody against antigen of Hantavirus can be screened out by phage display technique and then soluble scFv can be expressed in vector. Due to advantages of strong penetration and easy reaching target cell, soluble scFv is enabled extensive potent application in the research on diagnosis of Hantavirus and therapy and pathogenesis of HFRS. Therefore, in order to express soluble anti-NP scFv antibody and apply it in diagnosis of Hantavirus infection primarily, V_H gene and V_L gene were amplified by RT-PCR with template of total RNA extracted from peripheral lymphocyte of HFRS patients during the recovery stage. The scFv gene was constructed by SOE PCR from Vngene and V_L gene, and then ligated into T7 phage vector. The specific phage antibody was panned with expressed 76-118 nucleocapsid protein (NP)of hantaan. After amplified from positive clone, the scFv gene was cloned into pGEX-6p-l vector to express soluble scFv. Double scFv sandwich ELISA reagent was prepared with soluble scFv to detect NP antigen in sera as primary application of soluble scFv to establish a sensitive and specific diagnosis method for HFRS.
Method
一、Synthesis of scFv gene
The peripheral lymphocyte of HFRS patients during the recovery stage was separated by lymphocyte separation medium. Total RNA was extracted with RNAout. The first strands of cDNA of V_H gene and V_L gene were synthesized by reverse transcription assay with primer of oligo (dT) and template of total RNA.
The degenerate primers of V_H gene and V_L gene were designed and synthesized with EcoR I site being added into 5' terminal primer of V_H gene and HindIII site being added into the antisense primer of V_L gene. After V_H gene and V_L gene were amplified by PCR, scFv gene was constructed by SOE PCR with template of V_H gene and V_L gene.
二、Construction of T7 phage library
After being purified , scFv gene was digested with EcoR I and Hind III, and then analyzed by electrophoresis and purified again. The digested scFv gene was ligated with 2 arms of T7 Select10-3b digested with EcoR I and Hind III by T_4DNA ligase. The ligation product was packaged with T7 packaging extract. Packaging efficiency and scFv gene inserting rate were detected after plaque assay. The recombinant T7 phage was propagated in host BLT5403. The titer of the phage library was detected by plaque assay.
三、Panning of T 7 phage library
The ELISA plate was coated with recombinant NP antigen of Hantavirus 76-118 and blocked with BSA. After applied with T7 phage library and incubated for 30 min at room temperature, the well was washed 5 times with TBST and then bound phage was eluted with 1%SDS. The eluted phage was next round phage library that can be applied for next round screening after propagation in host BLT5403. The titter of next round phage library was calculated and the multiplicity of each round was calculated according to input and output of library. Above operation was repeated 4 times.
四、Identification of individual clones by enzyme immunoassay
After panning, the library was detected by plaque assay. After plaques were scraped at random, individual clones were propagated in BLT5403, and then purified with NaCl and PEG 8000. Phage antibody and peroxidase horseradish (HRP) were conjugated by 1% glutaraldehyde and then 10% superfluous glycine was added to block superfluous glutaraldehyde. At same time, wild type phage and peroxidase horseradish (HRP) were conjugated to serve as negative control. The HRP-conjugated phage antibody and negative control were applied to ELISA plate coated with NP antigens G1 glycoprotein and G2 glycoprotein of Hantavirus. After incubated for 1 hour at 37℃, the wells were washed 5 times with TBST. The substrates of o-Diaminobenzene and H_2O_2 were applied into each well, and after incubating for 15min at 37℃, 2M H_2SO_4 was added to terminate reaction. OD of each well was detected at 492nm wave length. If specimen A_(492)/ negative control A_(492) was above 2, the specimen was positive.
五、Construction of recombinant pGEX-6P-1- scFv
The phage antibody with highest activity was propagated in BLT5403, and T7 DNA was extracted with phenol-chloroform, and then scFv gene in T7 DNA was sequenced. The degenerate primers of scFv gene were designed and synthesized with BamH I site being added into 5' terminal primer and Sal I site being added into 3' terminal primer. The scFv gene was amplified by PCR with a template of T7 DNA and two primers of synthesized degenerate primers. The scFv gene of PCR product was purified. scFv gene and pGEX-6p-1 vector were digested with BamH I and Sal I , and analyzed by electrophoresis and purified. The digested scFv gene and large fragment of pGEX-6p-1 were ligated by T_4DNA ligase at 16℃for 4 h. The recombinant plasmid was transformed into competent E.coliBL-21 (DE3) and then cultured in Amp plate medium at 37℃overnight.
六、Screening and identification of recombinant plasmid
After being scraped, the individual clones were cultivated in Amp LB liquid medium. Recombinant plasmid was extracted by alkali lysis, and then identified by 0.7% agarose gel electrophoresis to find out candidate of positive clone. The candidate of positive clone was identified by digestion of BamH I and Sal I and PCR amplification. scFv gene in recombinant plasmid was sequenced.
七、Expression and purification of fusion scFv and identificationof scFv by SDS-PAGE and enzyme immunoassay
E.coliBL-21(DE3) ,which contained the pGEX-6P-1-NP-scFv, was cultivated in LB liquid medium supplemented with 50μg of ampicillin per ml. Culture was induced to produce fusion scFv by addition of IPTG to 1mM. After inducement, the culture was centrifuged to collect bacterial cells. The bacterial cells were re-suspended in PBS and lysed by ultrasonication. The fusion scFv was purified by a GST affinity chromatography column and was identified by SDS-PAGE.The soluble scFv was identified by enzyme immunoassay according to step四、.
八、Preparation of HRP-labeled scFv and Detection of sera specimen
The purified soluble scFv and HRP were conjugated by 1% glutaraldehyde and then condensed by PEG. ELISA plate was coated with soluble scFv and was blocked with BSA. Each well was applied with 50μl serum specimen. After incubated for 1 h at 37℃and washed 5 time with PBST , each well was applied with 100μl HRP-labeled scFv, and the well was washed 5 times with TBST. Following operation was as same as step四.
Result
一、Synthesis of scFv gene
1.5×10~7 peripheral lymphocyte of HFRS patients during the recovery stage was separated by lymphocyte separation medium. After being extracted with RNAout, the total RNA was identified by electrophoresis and 3 bands of 28 S RNA 18S RNA and 5 S RNA were obvious, and A_(260)nm/ A_(280)nm ratio was 1.8. These data indicated that integrity and purity of total RNA was very fine. After synthesized by RT- PCR , V_H gene and V_L gene were identified by electrophoresis, and the molecular weight (MW) of V_H gene and V_L gene was about 400 bp similar to the data in theory. After synthesized by SOE PCR by splicing V_H gene and V_L together, scFv gene was identified by electrophoresis and MW of scFv gene was about 750 bp similar to the data in theory.
二、Construction of T7 phage library
After construction of T7 phage library, efficiency of package was 1.5×10~7/μg DNA, and inserting rate of scFv gene was 90%, and the size of the library was 1.35×10~7, and titer of the primary phage library was 2.12×10~(10)PFU/mL.
三、Panning of T 7 phage library
The library was panned by 4 rounds of " absorption-elution-propagation" with NP antigen and the phage was enriched by 60 times.
四、Identification of individual clones by enzyme immunoassay
After the forth round screening, 23 individual clones were conjugated with HRP, and applied into ELISA plate coated with NP G1 glycoprotein and G2 glycoprotein of Hantavirus. Enzyme immunoassay shows that 19 clones with ability of specific binding to NP were identified and 2 of them had high ability of specific binding to NP antigen.
五、Construction of recombinant pGEX-6P-1- scFv
The phage antibody with highest activity was propagated in BLT5403. T7 phage DNA was extracted and after drying in air dissolved in TE buffer. T7 DNA was identified by 1% agarose gel electrophoresis and 36 kb T7 DNA was found. Sequencing demonstrated that scFv was gained. scFv gene was amplified by PCR with a template of T7 DNA, and scFv gene was identified by 0.7% agarose gel electrophoresis and 750bp band was found similar to the data in expectation.
六、Screening and identification of recombinant plasmid
Recombinant plasmid extracted by alkali lysis was identified by 0.7% agarose gel electrophoresis to find out candidate of positive clone. The candidate of positive clone was digested with BamH I and Sal I and identified by 0.7% agarose gel electrophoresis and 2 band of 5.0kb and 750bp was found. The result indicated that recombinant pGEX-6P-1- scFv was constructed successfully. At same time, scFv gene was amplified by PCR, and identified by 1% agarose gel electrophoresis and 750bp band was found. The data show that recombinant plasmid, which contained scFv, was constructed successfully. Sequencing showed scFv in recombinant plasmid was as same as scFv in T7 DNA.
七、Expression and purification of fusion scFv and identificationof scFv by SDS-PAGE and enzyme immunoassay
E.coliBL-21(DE3) ,which contained the pGEX-6P-1-NP-scFv, was cultivated in LB and induced to produce fusion scFv by addition of IPTG. The bacterial cells were lysed by ultrasonication and the fusion scFv was purified by a GST affinity chromatography column. The purified fusion scFv was identified by 10% SDS-PAGE electrophoresis and result show a 56Kda fusion scFv was found similar to expectation. Enzyme immunoassay shows that fusion scFv had ability of specific binding to NP antigen.
八、Preparation of HRP-labeled scFv and Detection of seraspecimen
The purified soluble scFv and HRP were conjugated by 1% glutaraldehyde and HRP-labeled scFv was condensed to 1ml by PEG. 56 specimens of sera, of which 29 sera were positive, of HFRS patients were detected by double scFv sandwich and positive rate was 51.79%. 66 specimens of sera in the control group were all negative.
Discussion
A phage display library was constructed with size of 1 .35×10~7, and inserting rate of scFv gene was 90%, and efficiency of package was 1.5×10~7/μg DNA, The titer of the primary phage library was 2.12×10~(10)PFU/mL. After 4 rounds of "absorption-elution-propagation" , the phage was enriched by 60 times. 2 clones with high ability of specific binding to NP antigen were identified by enzyme immunoassay. All the data were similar to the data in other researches conducted by Yao Longquan and others.
One of T7 phage's advantage is that it can display a very big exogenous protein and the biggest exogenous protein displayed by T7 phage is about 1200 amino acid while M13 phage can't. Other advantage of T7 phage is that it is so stable that it can't be ruined in severe condition, on the contrary, many other kinds of phages were inactivated during panning. Finally, T7 phage grows so quickly that plaque can be formed in only 3 hours, and this leads to reducing research time terrifically.
A pair of degenerate primers of V_H gene were designed to amplify most V_H gene and a pair of degenerate primers of V_L gene were designed to amplify most V_L gene. Not only antibody genes were amplified as more various as possible but also less primer was used, so that operation was simplified and time was saved. At same time, a sequence of gene of linkage peptide (Gly_4Ser)_3 was added into carboxylic terminal of V_H gene and amino terminal of V_L gene. V_H gene and V_L gene was joined directly to be scFv by SOE PCR as V_H -(Gly_4Ser)_3- V_L so the manipulation of gene was simplified greatly.
Now most research on phage display library ended after gaining corresponding antibody by panning. In this research, scFv gene was cloned into pGEX-6P-1 and soluble fusion scFv, which can be applied finally in following research, was expressed. Molecular weight of fusion scFv was about 56Kda,including scFv of 30Kda and vector protein of 26Kda. The vector protein normally did not affect the function of scFv, on the contrary, it is useful for purification of fusion scFv and it can be cut off by protease if necessary.
One of its biggest advantages of double scFv sandwich lied in its less cost compared with traditional double antibody sandwich. Compared with production of monoclonal antibody in cell by hybridoma technique, production of scFv in E. coli by gene-engineering can save more time and money, and is easier to operate.
56 sera specimen of HFRS patients and 66 sera in control groups were detected by double scFv sandwich. 29 sera from 56 sera specimen of HFRS patients were positive and positive rate was 51.79%, and all sera in control groups were negative. It is believed that patients are in viremia during early stage of HFRS and virus is gradually removed by immune system during the late stage of HFRS, so it is not easy to detected virus antigen in sera during the late stage of HFRS. So it was assumed that the 25 sera, which were negative in detection, of HFRS patients belong to patients during late stage of HFRS.
Conclusion
In this research, a T7 phage display library of human scFv was constructed with the size of 1.35×10~7, and the titer of the primary library was 2.12×10~(10)PFU/mL. 2 clones with high ability of specific binding to NP antigen were identified by enzyme immunoassay. In this research, recombinant vector pGEX-6P-1- scFv, which can express fusion protein, was constructed successfully. Soluble scFv was expressed efficiently and enzyme immunoassay showed that the soluble scFv has high ability of specific binding to NP antigen. In this research, the double scFv sandwich ELISA reagent was prepared first time with anti-NP scFv expressed in E. coli, and was applied to diagnose Hantavirus infection successfully.
引文
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