Acquisition and bioactivity analysis of single-chain fragment variable antibody against CSFV

Background Classical swine fever (CSF) is a highly contagious disease that threatens the pig industry. Passive immunization with neutralizing antibodies against classical swine fever virus (CSFV) is an effective prevention and therapeutic measure. Results In this study, to prepare a single-chain fragment variable (scFv) antibody against CSFV, two weanling piglets were injected twice with a CSFV attenuated vaccine. After the second immunization, peripheral blood lymphocytes were separated from the blood of the piglets by lymphocyte separation medium. After FACS, B lymphocytes with FITC labelled-E2 and FITC goat anti-swine IgG were sorted, extracted and cultivated on 96-well plates for 72 h. Then, the culture supernatants of B lymphocytes were screened by indirect ELISA with the purified CSFV-E2 antigen,and the target scFv antibody was bound to CSFV and obtained. Then, the scFv gene was inserted into the eukaryotic expression plasmid pCAGGS, and the constituted plasmid pCAGGS-scFv was transfected into ST cells. Western blot analysis showed that an approximately 31 kDa fusion protein was detected in the cell supernatant. Addition, the neutralizing capacity was measured in vitro. Indirect immunofluorescence assay (IFA) results showed that scFv-16 was able to neutralize CSFV. Conclusion Our data demonstrated that scFv-16 would be an effective diagnostic tool and potential therapeutic reagent for the CSFV infection in swine. Equal amounts of protein were separated on SDS-PAGE gels and transferred to nitrocellulose membranes. These membranes were blocked with 5% skimmed milk for 1.5 h at 37 ℃ and incubated with primary antibody and appropriate secondary antibodies. Signals were detected using a Supersignal West Pico Kit. Then the verified proteins were purified by concentrated column for further use. and evaluating the protective effect of the purified scFv-16 protein from the recombinant eukaryotic expression plasmid.

of pork. Therefore, the control and therapy of this disease is very important. Vaccination is a very effective way to prevent viruses and bacteria. In the process of antibody recognizing and neutralizing antigen, the "lock-key" relationship is formed by the binding of the antibody to the epitope on the surface of the Antigen [7,8]. So the structure of antigen and antibody recognition is very important.
The Fv fragment is the smallest unit of immunoglobulin molecule with antigen-binding activities [9]. An antibody in a scFv (single chain fragment variable) format consists of variable regions of heavy (VH) and light (VL) chains, which are joined by a flexible peptide linker and can be easily expressed by E.coli in their by functional form. Protein engineering has improved scFv properties, such as increased of affinities and alterations in the specificity [7,10].
The CSFV vaccine cannot arise from an ineffective neutralizing antibody as it cannot block the transmission of CSFV, thus leading to immune failure. Therefore, the purpose of this study is to develop a method that can rapidly generate neutralizing antibodies rapidly and improve the effective immune level to provide a new means for the prevention of CSFV. This study provides a new view on the prevention and treatment of animal diseases and provides technical information for the development of animal vaccinology.

Virus and cell culture
The CSFV strain was maintained in our laboratory. Swine testicle (ST) cells were routinely cultured in DMEM, supplemented with 10% fetal bovine serum (FBS) and propagated in a humidified atmosphere containing 5% CO 2 at 37℃.

Animal immunization
All animal experiments were performed in accordance with the guide for the Institutional Animal Care and Use Committee at Centers for Disease Control and Prevention (CDC) and in a facility accredited by the Laboratory Animal Care International. Two weanling piglets (thirty-days old) were purchased from CSFV seronegative pregnant sows at a local breeding farm and housed in separate sterile isolators and fed with commercial fodder and water. injected twice with CSFV attenuated vaccine.
After the second immunization, peripheral blood lymphocytes (PBLs) were separated from the blood of the pigs by a lymphocyte separation medium. After this experiment completed, all animals were euthanized humanely using sodium pentoarbiital anesthesis to reduce suffering.

B lymphocyte sorting
An equal amount of anticoagulation and lymphocyte separation fluid were gently mixed and horizontally centrifuged at 4000 rpm for 20 min. Then, lymphocytes in the middle layer were gently pipetted into a new microtube and washed with PBS. Finally, the cell pellet was dissolved in 0.5 ml PBS. After FACS B lymphocytes positive for FITC-E2 and FITC-IgG (sheep anti-swine) were sorted, extracted, and cultivated on 96-well plate for 72 h.

Generation of the VH and VL chains
According to the sequences of the pig antibody coding for the variable regions of the heavy chain(AF064688.1; AF064687.1; AF064690.1; and AF064689.1) and light chain (GQ867595.1; KF561242.1; and GQ867594.1) that were entered into GenBank, primers were designed to amplify the light and heavy chains of the CSFV antibody(listed in Table 1).Primers were synthesized by Shanghai Biotechnology Service Co., Ltd. Obtaining purified pCAGGS-scFv-16 After transfecting the pCAGGS-scFv-16 plasmid into ST cells, the cellular supernatants were collected after 48 h. Then the expression of the pCAGGS-scFv-16 protein was verified by Western blotting.
Equal amounts of protein were separated on SDS-PAGE gels and transferred to nitrocellulose membranes. These membranes were blocked with 5% skimmed milk for 1.5 h at 37 ℃ and incubated with primary antibody and appropriate secondary antibodies. Signals were detected using a Supersignal West Pico Kit. Then the verified proteins were purified by concentrated column for further use.

Neutralization Test
The purified pCAGGS-scFv-16 protein was mixed with 100 TCID50CSFV at a 1:1 ratio at 37℃ for 1 h, and the mixtures were added to ST cells. After 1 h of incubation, the ST cells were washed twice with PBS and then cultured for 48 h. Then, the neutralization effect of the purified pCAGGS-scFv protein was detected by indirect immunofluorescence. That is, the anti-CSFV-E2 was the primary antibody that was incubated with ST cell for 1.

Screening of IgG + B Lymphocytes
After separated from the pig peripheral blood, 2 µl anti-pig FITC-IgG and 2 µl FITC-E2 antibodies were added and incubated with 2*10 6 lymphocytes for 21 min at room temperature.Then the mixture was centrifuged at

Construction of Single Chain Antibody
Total RNA from single-chain antibody-positive cytosol was extracted by an RNA extraction kit, and reverse transcription was used to prepare the cDNA. The VH and VL genes were amplified using the RNA extraction kit.
VH-Linker-VL fragments were constructed by SOE-PCR. As shown in Fig. 4A, PCR amplified an approximately 730 bp band, which is correct and expect size of the fragment (Figs. 2B and 2C).

Sequencing analysis of single chain antibody positive clones
The positive clones were connected to the T vector and then sequenced and analyzed. The results were as follows: compared with the single chain Fv antibody published in the NCBI database, the 5 McAbs obtained from pigs were also single chain Fv antibodies. These include the largest variation in the CDR region, less variation in the FR region, and the largest change of amino acids in the CDR3 region of heavy and light chains (Fig. 3).

Construction of a eukaryotic expression vector
As shown in Fig. 4B, the pCAGGS vector and scFv genes were simultaneously digested by Nhel and EcoRI restriction endonucleases. The corresponding fragments were recovered and ligated. The correct recombinant cloning plasmid pCAGGS-scFv was screened by plasmid extraction.
Evaluation of the in vitro cytotoxicity of scFv-16 The MTT assay was performed to investigate the possible cytotoxicity of pCAGGS-scFv-16 toward ST cell growth.
The MTT assay showed no cytotoxic effects toward ST cells, suggesting that pCAGGS-scFv-16 was also safe for ST cells.

The expression of scFv-16 in ST cells
The expression of pCAGGS-scFv in ST cells was analyzed by Western blotting. Cells transfected with an empty vector were used as a control. 24 h, 36 h,48 h, 60 h, the cell lysates were collected, and these samples were subsequently identified by means of protein gel electrophoresis. The CSFV serum was incubated with primary antibody, followed by incubation with secondary antibody (HRP sheep anti-swine). As shown in Fig. 2D, successful transfection and effective expression of pCAGGS-scFv were confirmed by Western blotting.

Viral Neutralizationassay
The purified scFv was mixed with CSFV, and the neutralization index was detected in ST cells.

Discussion
Classical swine fever (CSF) is a highly contagious disease caused by the classical swine fever virus (CSFV) [11].
The high incidence and mortaligy of CSF threatens the pig industry, and CSF is an acute and highly infectious disease among farmed animal industries worldwide [12,13]. Vaccination against CSFV has controlled the epidemic of classical swine fever to a certain extent. However, because of the immune suppression caused by CSFV, the level of effective neutralized antibodies caused by the vaccine is low, so immunization cannot adequately prevent a CSF epidemic.
As an important tool in modern life science research, the monoclonal antibody technique covers the hybridoma technique, phage display technology, single B cell antibody preparation technique and so on [14]. Antibodies prepared by the hybridoma technique have high immunogenicity and a short half-life period, which leads to an unsatisfactory clinical efficacy [15]. Because of its simplicity and convenience, phage display technology is the most widely used method in antibody preparation. However, due to the random assortment of heavy and light chains, the native mating of heavy and light chains in antibodies cannot be maintained [16]. Currently, as an emerging technology, single B cell antibody preparation is a technique that clones and expresses single antigenspecific B cell antibody genes in vitro. This technique reserves the native mating of heavy and light chains, and has the advantages of good genetic diversity, high efficiency and low cell requirement [17]. Therefore, in this study, we used a B cell antibody was used for focus to acquire a single-chain antibody to CSFV.
A neutralizing antibody is an important factor in antiviral immunity and is also a key index to analyze the immune effect of vaccines [18]. Neutralizing antibodies kill viruses by neutralizing infectious virus particles, thus preventing viral infection and eliminating pathogens [19]. Currently, the vaccine is mainly developed by selecting protective antigens and inducing immune neutralizing antibodies to neutralize a viral infection. However, the protective antigens of viruses that can induce immunosuppression often fail to induce effective neutralizing antibodies, resulting in an unsatisfactory immune response. Therefore, designing the design of vaccines that can induce highly effective neutralizing antibodies has attracted more attention. Research on neutralizing antibodies in humans has developed rapidly, especially in the fields of AIDS, hepatitis A and hepatitis B. The current method is to establish an antibody gene library through a phage vector expression system, especially the pCom3 expression system, to screen and obtain anti-viral neutralizing and monoclonal antibodies, which can directly prepare antibodies or antibody vaccines in vitro for emergency immunoprophylaxis and treatment. Currently, human monoclonal antibodies against influenza virus, anti-HbsAg, anti-respiratory syncytial virus (RSV) F protein, anti-herpes simplex virus, anti-HIV gp120 and anti-hepatitis C virus have been successfully screened by this technology [20][21][22][23][24].
Currently, the idea of animal vaccine research is to express protective antigens of pathogens and to stimulate the body to produce antibodies. However, only some of these antibodies have a neutralizing viral activity, and can be identified as neutralizing antibodies, addition, the remaining antibodies have no neutralizing activity. Therefore, although the vaccine can induce a high level of antibodies, the immune effect is not ideal. In addition, pathogens with an immunosuppressive function can inhibit immunity. In that case, the immune system cannot arouse an effective immune response to eliminate pathogens, so the protective antigen cannot be used as an ideal vaccine to obtain an effective immune reaction. In

Ethics approval and consent to participate
This study was approved by the Institutional Animal Care and use committee of Shanghai Academy of Agricultural Science. No specific permissions were required for the collection of samples. All sampling and publication of the data were approved by the farm owners.

Consent for publication
Not applicable

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Acknowledgement
We would like to thank AJE company for carefully revising the manuscript. Arrivechecklist.docx