| BackgroundStreptococcus mutans(SM)is a type of Gram-positive facultative anaerobic bacteria with cell wall polysaccharide belonging to Streptococci.The SM is predominant pathogen causing the dental caries It infects tooth via its surface protein SAⅠ/Ⅱ adhering to the biofilm on tooth surface.Upon administrating sucrose-rich food,the bacteria consumes the sucrose producing acidic products,which lead to the destruction of enamel leading to the occurrence of caries.Caries is an irreversible injury on teeth,which are occurred in 70%children under 5 years old and 80%in adults.Therefore,prevention and treatment of SM infection is a promising strategy for reducing the incidence of dental caries Passive immunotherapy has been widely used in the treatment and prevention of infectious diseases through administrating the therapeutic immunoglobulin or engineering antibodies.The antibodies inhibit the pathogen via neutralization or ADCC effect.The previous reports from other laboratories indicated that passively delivering of antibodies against SM could significantly reduce the incidence of SM infection and caries.The SM surface protein SAⅠ/Ⅱ and glucosyltransferase are two virulence that are responsive for SM adhering to and forming biofilm on tooth surface.The antibodies against SAⅠ/Ⅱ have been showed to prevent the caries in animals models and volunteers.There are divergent vehicles used for antibody production,e.g,sugar cane,tobacco,yeast and lactobacillus.Most of the recombinant IgG are from mouse IgG1,which limited its application clinically due to its immunogen and difficulty in quality control.The engineering single-chain antibody on surface of lactobacillus casei(a food safety probiotic bacteria)was reported to prevent the SM infection and reduce the incidence of tooth caries in rat models However,this tremendous product could not be used in clinic due to carrying the antibiotics resistant genes in the recombinant vectorThe single chain antibody(ScFv),consisits of light chain and heavy chain variable regions.It has been widely used in the diagnosis and the treatment of diseases with distinguished characteristics,e.g,small molecular weight,low antigen activity,easily operation and expression.Our previous report indicated that the ScFv to SAⅠ/Ⅱ expressed on the surface of lactobacilli can effectively reduce the caries induced by SM in rat model.However,the expressing vector,which contains kanamycin and erythromycin resistant gene encoding limited its application.In this project,we will establish a ScFv-phage library to identify high affinity ScFv against SAⅠ/Ⅱ,and express ScFv-Fc recombinant antibodies via CHO(Chinese hamster overy)expression system,and the biological activity of ScFv-Fc against SM infection will be studied here.Phage display technology(PDT)is molecular technology to express and display the single chain antibody or polypeptide protein on the surface of phage,and its corresponding gene sequence will appear in its nucleic acid.PDT has been widely used for novel antibody identification and antibody engineering.CHO cells is a unique golden hallmark cell line,which has been used to express and produce the recombinant proteins or antibodies in industry.The number of therapeutic antibodies expressed via CHO have been used clinically,such as avastin,anti-PD-1 antibody et al.There are a number of fusion protein of ScFv antibody and Fc-fragment were expressed in CHO cells for pathogen therapy,e.g.ScFv-Fc for HBV.In this project,our goal is to express a therapeutic effect of ScFv-Fc against SM-SAⅠ/Ⅱ via CHO cells.PurposeTo acquire the high affinity ScFv-Fc fusion antibody against SAⅠ/Ⅱ protein via the technologies,including computer predication model,molecular cloning,ScFv phage display library and CHO expression system.Methods(1)The amino acid sequence of ScFv from mouse against SAⅠ/Ⅱ were analyzed via computer predication software to predict the key amino acid in ScFv.The ScFv mutation sequence was amplified by Overlap PCR,and then cloned into phage display plasmid to construct phage display library.(2)Four rounds of "adsorption-elution-amplification" in vitro biological panning of phage ScFv library was carried out by PDT,and the positive phage clones were screened out,and non-specific binding phage clones were verified by bacterial PCR.(3)The phage positive clones were detected by Phage-ELISA,eight representative ScFv cDNA clones were selected after sequence analysis and subcloned into eukaryotic expression vector PHB-Fc,and then transfected into HEK293T cells to detect its expression.(4)The recombinant plasmids PHB-ScFv-Fc were transfected into CHO-K1 cell line,and the binding activity to SAI/II protein in the supernatant was detected by ELISA.(5)The high binding activity CHO-K1 cell lines were expanded to culture,and the supernatant was collected.The ScFv-Fc recombinant antibodies with high purity and concentration were purified by HiTrap protein affinity chromatography.(6)The binding activity of the three purified ScFv-Fc recombinant antibodies to SAⅠ/Ⅱ was detected by ELISA.(7)The specifically bingding of three ScFv-Fc recombinant antibodies to the SAⅠ/Ⅱ of SM was detected by Western blotting and immunofluorescence(8)The affinity of three ScFv-Fc recombinant antibodies to SAⅠ/Ⅱ was detected by non competitive ELISA.(9)The agglutination of three ScFv-Fc recombinant antibodies to SM was detected by agglutination test.(10)The effect of three Scfv-Fc recombinant antibodies on the formation of SM biofilm was detected by biofilm formation test.Results(1)The phage ScFv library of 106 was successfully constructed by computer predication software and molecular cloning technology.(2)192 phage positive clones were screened by PDT,and 72 clones were identified by bacterial PCR.53 clones were randomly selected from 72 positive clones for Phage-ELISA,and 36 clones were sequenced correctly.After multiple sequence aligment,8 representative clones were selected and subcloned into PHB-Fc vector.(3)The 293T and CHO-K1 cell lines were successfully constructed,and three ScFv-Fc recombinant antibodies were expressed and purified,which had strong binding activity to SAⅠ/Ⅱ protein.(4)Western blot and immunofluorescence showed that the three ScFv-Fc recombinnant antibodies could specifically bind to the SAⅠ/Ⅱ protein of SM.(5)Non competitive ELISA showed that the affinity of three ScFv-Fc recombinant antibodies to SAⅠ/Ⅱ protein was 2 fold more than wild type of ScFv-Fc.(6)The agglutination assay showed that recombined ScFv-Fc can neutralize and agglutinate SM.(7)Three kinds of ScFv-Fc recombinant antibodies have a weak inhibitory effect on SM biofilm formation.Conclusions(1)The affinity between ScFv-Fc and SM surface protein SAI/II has been successfully improved via computer predicting,molecular cloning,phage mutation ScFv display,and CHO eukaryotic expression,which affinity is 2 fold more than wild-type ScFv.(2)Three high affinity CHO-K1 cell lines expressing ScFv-Fc recombinant antibodies were obtained.(3)The novel ScFv-Fc reconbinant antibodies could interact with and agglutinate SM and weakly inhibit biofilm formation in vitro. |
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