The Preparation Of Single-Chain Variable Region Fragments Against Stapylococcus Aureus And Its Therapeutic Roles In Bovine Mastitis

Bovine mastitis is one of the most common diseases affecting the dairy industry,and causes substantial economic losses due to reduced milk production.Although various pathogens can cause mastitis,Staphylococcus aureus is one of the most common causative agents.Antibiotics are primarily used to treat S.aureus-induced mastitis.However,the emergence of antibiotic-resistant strains of S.aureus can reduce the effectiveness of such treatments,and antibiotic-contaminated milk may be harmful to human health.In recent years,Many vaccines have been produced in an attempt to elicit antibodies against adhesion factors,enzymes,toxoids,and capsule polysaccharides of S.aureus,However,these vaccines have not provided adequate protection against S.aureus infection in cattle.Along with the development of molecular biology and immunology technology,antibody therapy may represent an alternative strategy for the prevention of bovine mastitis.The single-chain variable region fragment?scFv?is composed of the variable regions of the heavy?VH?and light?VL?chains of an immunoglobulin molecule,which are joined together by a flexible peptide linker and possesses the complete antigen-binding site.As one popular type of genetically engineered antibody,scFv has such properties as low molecular weight,powerful penetrability and low immunogenicity,relative to that of whole immunoglobulin molecules.The absence of the Fc enables the use of scFv in vertebrates because it avoids the negative effects of Fc-dependent host immune responses,which may be undesirable for some applications.In addiation,more and more scFv-based drugs are in treament of common diseases on clinical.In view of this,we constructed bovine scFvs against S.aureus and evaluated the protective effects of these scFvs.Our results provide a foundation for the development of scFv-based therapeutics for the prevention and treatment of bovine mastitis.1 Construction of bovine scFv that bind fibronectin-binding protein A and clumping factor A of Stapylococcus aureusIn generating the bovine scFv,one key step was designed the primers to amply VH and VL domains of antibodies.However,there have been no reports of bovine scFv so far.In this chapter,we designed a pair of primers through analysising of bovine antibody gene clues in the database.Total cellular RNA was isolated from the peripheral blood lymphocytes of cows with S.aureus-induced mastitis,and first-strand cDNA was synthesized using a reverse transcription kit.VH and VL gene were amplified from cDNA by PCR and the scFv was spliced with a DNA sequence encoding a?Gly₄-Ser?₃ peptide linker in an overlap PCR.The scFv gene was cloned into a pOPE101-XP vector and expressed in E.coli XL1-Blue,meantime soluble scFv in periplasm was extracted.The scFvs were selected based on the binding of fibronectin-binding protein A?FnBPA?and clumping factor A?ClfA?,which are considered to be the most important adhesion factors involved in the pathogenesis of mastitis using an indirect ELISA,and two scFvs,designated as ZW.28 and ZW.132,were selected(OD₄₅₀?0.25).The Western blot analysis showed that both scFvs bound FnBPA and ClfA specifically.And the two scFvs can inhibited the growth of S.aureus in culture medium to a certain degree.In this chapter,it not only confirm the primer that code the bovine scFv gene to provide a foundation for the construction of the bovine scFv phage display library;but also certify the feasibility of bovine scFvs can inhibited the growth of S.aureus,this offers proof of principle of the development and functional study of bovine scFv.2 Construction of bovine scFv phage display library and screening of scFvs against Stapylococcus aureusWe constructed the bovine scFv phage display library through the primers of coding bovine scFv gene that were identified in the last chapter.Total cellular RNA was isolated from the peripheral blood lymphocytes of cows with S.aureus-induced mastitis,and first-strand cDNA was synthesized using a reverse transcription kit.VH and VL gene were amplified from cDNA by PCR and the scFv was spliced with a DNA sequence encoding a?Gly₄-Ser?₃ peptide linker in an overlap PCR.The scFv gene was cloned into the pCANTAB-5E phagemid and the ligation mixture was used to transformed electrocompetent TG1 E.coli by electroporation.The phage display scFv library had a titer of 2×10⁶cfu/mL after fifty transformations were performed.The positive rate of the library was 90%and the diversity was acceptable.The supernatant of the S.aureus lysate was used as the antigen for panning and affinity selection.After four rounds of panning,the phages were isolated at a titer of10⁷cfu/mL,achieving an 81-fold enrichment of phage particles.Ninety-six phage clones were randomly selected to confirm their reactivity against the S.aureus using phage ELISA and eight phage clones had an OD₄₅₀>0.6,were designated as ZW.1,ZW.2,ZW.12,ZW.22,ZW.33,ZW.68,ZW.73,ZW.88,demonstrating high affinity.The sequencing results for eight scFvs showed that the sequences of the FRs were highly conserved,and that the sequences of the CDRs displayed significant diversity,especially in CDR3 of the VH domain.3 Anti-Staphylococcus aureus scFvs provide protection against mastitis in miceWe obtained eight scFvs that that bound S.aureus antigens with high affinity using phage display technology.However,pCANTAB-5E phagemid contained E-tag and there has been no purification method of E-tag by now.So the eight scFvs phagemid DNA ligated into the pET-28a?+?bacterial plasmid for expression and purification.The purified scFv?or cocktail of eight purified scFvs?combined with S.aureus?ATCC25923?in culture medium and we found that S.aureus growth in LB broth was inhibited to a certain degree by all eight of the selected scFvs and by a combined mixture of the eight scFvs.Interestingly,the scFvZW.12,ZW.88 and mixture of the eight scFvs were the most effective inhibitors of S.aureus growth,and the LB medium remained clear throughout the observation period.Previous studies in animal models have shown that a cocktail of neutralizing antibodies that recognize different epitopes provides more effective protection than a single antibody.Therefore,we hypothesized that the combination of the eight scFvs would likely provide more effective protection in vivo,compared with treatment using a single scFv.Twenty-four Kunming mice that had delivered their first litter of pups 10 to 15 days prior were randomly assigned to the control?C?,negative control?NC?,positive control?PC?,and scFv treatment groups for intramammary bacterial challenge.The control and NC groups were injected subcutaneously into the teats of the R4 and L4mammary glands of the mice with physiological saline.The PC and scFv groups were injected subcutaneously into the teats of the R4 and L4 mammary glands of the mice with penicillin or scFv,respectively.Six hours later,50?L of physiological saline was injected subcutaneously into the teats of the R4 and L4 mammary glands of the mice in the control group,and 50?L of 10⁸cfu/mL S.aureus?ATCC25923?was injected subcutaneously into the teats of the R4 and L4 mammary glands of the mice in the NC,PC,and scFv groups.The histopathological examination showed that treatment with these scFvs prior to bacterial challenge maintained the structure of the mammary acini,decreased the infiltration of polymorphonuclear neutrophils,increased the levels of interferon-gamma and interleukin-4?P<0.05?,and reduced the level of tumor necrosis factor-alpha in mammary tissues?P<0.05?,compared with those in mice that received the physiological saline treatment.These novel bovine scFvs may represent suitable candidates for therapeutic agents for the prevention of S.aureus-induced bovine mastitis.4 The effect of bovine scFvs gene therapy against Stapylococcus aureus infection in a mouse model of mastitisCompared with intact IgG,scFv has such properties as low molecular weight,powerful penetrability,and low immunogenicity,rendering scFv a powerful tool for drug targeting.However,scFv has a relatively short half-life and is cleared from the blood more rapidly than the parent immunoglobulin,which presents challenges for wide-spread use in disease therapy.We all know that gene encoded by a DNA expression vector can be readily detected following injection into mouse skeletal muscle in vivo and was present in the muscle for at least 2 months.In this chapter,ZW12 and ZW88 scFvs,which were found to be the most effective inhibitors of S.aureus growth in LB medium,were inserted into the eukaryotic vector pcDNA3.1 and transfected into COS-1 cells.The presence of pcDNA3.1-scFvs was then verified in cell lysate and cell culture supernatants.Kunming mice were administrated a mixture of pcDNA3.1-scFv12 and pcDNA3.1-scFv88 through the fourth mammary duct,which resulted in detectable pcDNA3.1-scFvs expression as early as 3 days postinoculation,and constant levels were maintained for 7 days to postinoculation day11.Thirty lactating postpartum Kunming mice were randomly assigned to a control?C?,negative control?NC?,positive control?PC?,scFv-low?SL?treatment,or scFv-high?SH?treatment group for intramammary bacterial challenge.On postpartum day 7,mice in the control and negative control groups were injected with100?g of pcDNA3.1 into the ducts of the fourth mammary glands.The SL and SH treatment groups were injected with 100?g and 200?g of pcDNA3.1-scFv12 and pcDNA3.1-scFv88,respectively,into the ducts of the fourth mammary glands.After 7days,the mice in the PC group received subcutaneous injection of penicillin into the fourth mammary glands.Six hours later,50?L of physiological saline was injected subcutaneously into the teats of the R4 and L4 mammary glands of the mice in the control group,and 50?L of 10⁸cfu/mL S.aureus?ATCC25923?was injected subcutaneously into the teats of the R4 and L4 mammary glands of the mice in the NC,PC,SL and SH groups.The histopathological examination showed SH treatment group prior to bacterial challenge maintained the structure of the mammary acini,decreased the infiltration of polymorphonuclear neutrophils,and reduced the level of interleukin-1?in mammary tissues,compared with those in mice that received the physiological saline treatment?P<0.05?.These results are expected to provide a foundation for the development of gene therapy strategies for the treatment of bovine mastitis.