| IntroductionHemorrhagic 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, VH gene and VL 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 VL 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 geneThe 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 VH gene and VL gene were synthesized by reverse transcription assay with primer of oligo (dT) and template of total RNA.The degenerate primers of VH gene and VL gene were designed and synthesized with EcoR I site being added into 5' terminal primer of VH gene and HindIII site being added into the antisense primer of VL gene. After VH gene and VL gene were amplified by PCR, scFv gene was constructed by SOE PCR with template of VH gene and VL gene.二,Construction of T7 phage libraryAfter 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 T4DNA 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 libraryThe 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.å››,Identification of individual clones by enzyme immunoassayAfter 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 H2O2 were applied into each well, and after incubating for 15min at 37℃, 2M H2SO4 was added to terminate reaction. OD of each well was detected at 492nm wave length. If specimen A492/ negative control A492 was above 2, the specimen was positive.五,Construction of recombinant pGEX-6P-1- scFvThe 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 T4DNA 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.å…,Screening and identification of recombinant plasmidAfter 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 immunoassayE.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 stepå››,.å…«,Preparation of HRP-labeled scFv and Detection of sera specimenThe 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 stepå››.Result一,Synthesis of scFv gene1.5×107 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 A260nm/ A280nm ratio was 1.8. These data indicated that integrity and purity of total RNA was very fine. After synthesized by RT- PCR , VH gene and VL gene were identified by electrophoresis, and the molecular weight (MW) of VH gene and VL gene was about 400 bp similar to the data in theory. After synthesized by SOE PCR by splicing VH gene and VL 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 libraryAfter construction of T7 phage library, efficiency of package was 1.5×107/μg DNA, and inserting rate of scFv gene was 90%, and the size of the library was 1.35×107, and titer of the primary phage library was 2.12×1010PFU/mL.三,Panning of T 7 phage libraryThe library was panned by 4 rounds of " absorption-elution-propagation" with NP antigen and the phage was enriched by 60 times.å››,Identification of individual clones by enzyme immunoassayAfter 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- scFvThe 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.å…,Screening and identification of recombinant plasmidRecombinant 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 immunoassayE.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.å…«,Preparation of HRP-labeled scFv and Detection of seraspecimenThe 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.DiscussionA phage display library was constructed with size of 1 .35×107, and inserting rate of scFv gene was 90%, and efficiency of package was 1.5×107/μg DNA, The titer of the primary phage library was 2.12×1010PFU/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 VH gene were designed to amplify most VH gene and a pair of degenerate primers of VL gene were designed to amplify most VL 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 (Gly4Ser)3 was added into carboxylic terminal of VH gene and amino terminal of VL gene. VH gene and VL gene was joined directly to be scFv by SOE PCR as VH -(Gly4Ser)3- VL 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.ConclusionIn this research, a T7 phage display library of human scFv was constructed with the size of 1.35×107, and the titer of the primary library was 2.12×1010PFU/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.
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