| Objective: Recognition and binding specificity of the antibody with the corresponding antigen,is an important means to remove the resist external invasion.Antibody for the corresponding antigen has the characteristics of high affinity and high specificity to make antibody in the diagnosis and treatment of disease has shown incomparable advantages of other types of drugs.Antibody drug has undergone monoclonal antibodies,Polyclonal antibodies,recombinant antibody in three stages.Polyclonal antibody,a hybrid antibody produced by the plurality of B cells,high affinity for the antigen,but poor specificity.In 1975 advent of hybridoma technology,people can produce excellent specific monoclonal antibodies.But the monoclonal antibody mainly from a murine,such heterologous making body rejection.In recent years,more research is genetically engineered antibody-recombinant antibodies,is produced using recombinant DNA and protein engineering technology to encoding the antibody genes according to the different needs of the transformation and restructuring of the antibody.Currently genetically engineered antibodies including chimeric antibodies,humanized antibodies,fully human antibodies.By gene manipulation,antibody forms may be obtained by unconventional techniques,and thus more suitable for the diagnosis and treatment of disease.In particular,the use of in vitro selection of fully human antibody,antibody drug has become the main trend.Staphylococcus aureus is a versatile pathogen and a common cause of nosocomial and communityacquired infections.The infection of multi-drug-resistant strains of Staphylococcus aureus,have a high mortality rate,it has become one of the world's three major problems the treatment of infections.Staphylococcus aureus ?-hemolysin(?-HL)is already recognized as the most important virulence factor toxin.In this experiment,expression and purification of soluble Staphylococcus aureus ?-HL as an antigen for the production of the anti-?-HL fully human antibody,in order to provide a new treatment of Staphylococcus aureus infection.Methods: The total RNA of Staphylococcus aureus was extracted and the cDNA of ?-HL was amplified by RT-PCR.The DNA of ?-HL and pCold-TF plasmid was digested and ligated by T4 ligase and then transformed into E.coli TOPO 10.The recombinant plasmid ?-HL/pCold-TF which verified by sequencing was transformed into E.coli BL21 for expression.The expression products was identified by SDS-PAGE and western blot.Biotinylated protein ?-HL-Antigen by using phage display technology on the natural antibody library was built in the early 3 rounds of enrichment,detected by ELISA technique selected from enriched single-chain antibody library full human single chain antibody(scFv)against ?-HL.For detected ?-HL fully human single-chain antibody positive clones were highly expressed and sequenced to verify its correctness,through a variety of bacterial binding reaction,detect ?-HL fully human single-chain antibody specificity by ELISA.Results:(1): The size of amplified cDNA of ?-HL was about 900 bp and the expressed soluble fusion protein of ?-HL was about 90 kDa(including the molecular chaperone in the vector)after inducing expression for 24 h at 15?.The western blot results showed that the expressed protein was the fusion protein of ?-HL.The purified ?-HL was injected into BABL/C mice for making antiserum.The results showed that the antiserum had good binding activity with Staphylococcus aureus and the titer was greater than 10,000×.(2): Biotinylated protein ?-HL-Antigen by using phage display technology on the natural antibody library was built in the early 3 rounds of enrichment,detected by ELISA technique selected from enriched single-chain antibody library full human single chain antibody(scFv)against ?-HL,select few positive clones,and the sequencing is correct.By combining a variety of bacteria,verify the specificity against Staphylococcus aureus by ELISA.Conclusion: The ?-HL of Staphylococcus aureus was successfully cloned and the soluble fusion protein of ?-HL was successfully expressed.Successfully screened to have a high specificity of full human scFv against ?-HL. |
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