Preparation Of Nanobodies Against The Surface Protein VirB12 Of Brucella Type ? Secretion System

Brucellaspp is a compatible intracellular parasitic gram-negative short bacillus,which infects a variety of tissues and organs and is the causative agent of zoonotic brucellosis(brucellosis).Animal brucellosis can lead to abortion and other diseases.Human brucellosis usually presents with high fever,undulating fever,and is often complicated by arthritis.It is harmful to animal husbandry,and its large area infection can lead to significant economic losses,so it is particularly important for the prevention and detection of brucellosis.At present,bacterial culture,isolation and identification,agglutination test,serological examination and other methods are mainly used for the detection of Brucella infection and vaccine effect,but these methods have the disadvantages of specificity and sensitivity,so it is necessary to develop detection methods and reagents with higher specificity.The Brucella type IV secretion system is a multiprotein complex that runs through the bacterial wall and outer membrane and is an important virulence factor of Brucella.Among the 12 proteins of Vir B(Vir B1-Vir B12),Vir B12 plays an important role in bacteria-host interaction in the Brucella type IV secretion system and can be used as a brucella serological detection marker antigen.Nanobody is a novel small molecule antibody derived from the camel heavy chain antibody variable region(VHH).It has the advantages of simple structure,easy modification and stable properties,and low production cost,and has good application potential in the fields of disease diagnosis and treatment,and pathogen detection.In this study,we screened nanobodies against Vir B12 by phage display technology and studied their properties to provide an experimental basis for the development of nanobody-based brucellosis detection methods in the future.The study is divided into the following two parts:1.Construction of VHH phage display library and screening and identification of Vir B12 nanobodiesXinjiang bactrian camels were immunized with Vir B12 recombinant protein for six times.Total RNA was extracted from lymphocytes isolated from peripheral blood and c DNA was obtained by reverse transcription.The VHH gene fragment was amplified by nested PCR with VHH specific primers.The obtained VHH fragments were ligated into the p MECS phage particle vector and electroporated into E.coli TG1 competent cells to form a VHH gene library.The capacity was calculated to be 2.8×108 cfu/m L based on the number of transformants.The positive rate of PCR was 100%.DNA sequencing of randomly selected clones showed that the constructed library had high library capacity and good diversity.M13K07 helper phage was used to infect the library TG1 bacteria to form the M13phage VHH display library.Anti-Vir B12 nanobodies were screened by ELISA solid-phase affinity enrichment method for a total of three rounds of enrichment.The clones enriched in the second and third rounds were randomly picked up,and the binding of soluble expressed nanobodies to Vir B12 was analyzed using monoclonal ELISA,and the OD450 nm ratio greater than 3 bound to the unrelated antigen BSA was judged as a positive clone.A total of 46 positive clones were obtained,and repetitive sequences were removed by sequence determination and multiple alignment,and finally five non-redundant sequences,named D1,E6,H8,H9 and H10,were obtained.These clones were shown to belong to five different families in the evolutionary analysis.2.Prokaryotic expression,affinity and thermal stability analysis of Vir B12nanobodiesIn order to obtain high expression,the five nanobody genes identified by screening were transformed into the WK6 strain for interstitial soluble expression at 16?,and the expressed recombinant protein was purified using Ni column affinity chromatography.The results showed that all five nanobodies could be expressed in the soluble form of WK6 bacteria,and the expression levels reached more than 45 mg/L.SDS-PAGE results showed that five anti-Vir B12 nanobodies D1,E6,H8,H9 and H10 with a purity of nearly 90%were obtained.Westernblot and indirect ELISA were used to detect the binding activity of the five nanobodies to the Vir B12 protein;thermal stability was treated with serial temperatures(4?,16?,22?,37?,50?,60?,70?,80?,and 90?),and ELISA was used to detect the antigen-binding activity retained by the nanobodies.The results showed that all five nanobodies had high antigen-binding activity and there was a significant antigen-antibody concentration dependence.Among them,D1 still had good antigen-binding activity at 0.0001?g/m L,indicating that the antibody was a high-affinity antibody.In the determination of stability,all four nanobodies showed high thermal stability and could still retain more than 60%of the binding activity after temperature treatment at 90?,of which the antigen-binding activity of D1 after 90?was basically unaffected,showing extremely high thermal stability.Among the five antibodies,H8 had the lowest thermal stability.In summary,in this study,a VHH phage display library with a library capacity of2.8×10~8cfu was constructed from Xinjiang bactrian camel lymphocytes immunized with Vir B12 recombinant protein,and five anti-Vir B12 nanobodies with high affinity were obtained by solid phase screening enrichment and soluble monoclonal ELISA assay identification,all of which could be highly expressed in bacteria and had high thermal stability,and these results laid the foundation for the further development of Vir B12 nanobodies.