Study On The Bispecific Antibody Based Rapid Diagnosis Of Tropical Diseases

Tropical infectious diseases are important illnesses that threaten soldiers' health in South China.Army officers and soldiers are lack of self-protection awareness.It is vulnerably infected when they enter a new environment due to lack of immunity.Our soldiers need to carry out a variety of military tasks,so we urgently need to establish perfect pathogen detection,diagnosis,prevention and related technologies,in dealing with the threat of tropical infectious diseases.Malaria is a insect-borne infectious diseases caused by plasmodium,widely distributed in tropical regions.It is one of the eight tropical diseases listed by the WHO.Especially during the recent years,emerging drug resistance bring a major challenge to global malaria control and elimination.Scrub typhus is a insect-borne infectious diseases caused by Orientia tsutsugamushi.And in recent years,the affected area has a spread trend in our country.As islands in the South China Sea is rich,warm and rainfall.A large part of the area has been proved to be the epidemic focus of scrub typhus.Because most of soldiers who garrison the islands are immigrant population,they are easy infected and then affect the completion of various tasks.Although not one of the eight tropical diseases listed by the WHO,it is a important reason for non-combat loss of members in the southern war zone.In order to establish a method that meet the need of fast,accurate and convenient diagnosis of tropical diseases,we introduce the bispecific antibodies.Genetic engineering bispecific antibodies(Bs Ab)comprise two different specificities,and can bind simultaneously to a specific pathogen antigen and a given detection moiety enables them to function as excellent bifunctional immunoprobes in diagnostic assays.We generate it by compose two different monoclonal antibodies' VH(variable domain of heavy chain)and VL(variable domain of light chain)domains using linkers such as non-covalent bond,covalent bond,leucine zipper,helix-turn-helix and so on.We construct an anti-red blood cell/anti-pathogen bispecific antibody.This recombinant antibody can combine human red blood cell and pathogen in the blood at the same time,and then the red blood cells agglutination occur.In this way,we can detect pathogen infection rapidly,conveniently and accurately.In this study,we first isolated mRNA from hybridoma cells which secrete anti-merozoite surface protein of malaria monoclonal antibody,anti-outer membrane protein of Orientia tsutsugamushi monoclonal antibody,and anti-red blood cell membrane nonagglutination protein monoclonal antibody.Then reverse transcript of c DNA which served as templates for monoclonal antibody VH and VL genes amplification.We constructed anti-merozoite surface protein of malaria sc Fv by consisting of VH and VL,which were joined together by a flexible peptide linker using overlapping PCR.We constructed anti-outer membrane protein of Orientia tsutsugamushi /anti-red blood cell membrane nonagglutination protein Bs Ab by consisting of anti-surface membrane protein of Orientia tsutsugamushi monoclonal antibody VH and VL,and anti-red blood cell membrane nonagglutination protein monoclonal antibody VH and VL,which were joined together by two short flexible peptide linkers L10(SAKTTPKLGG)and a long flexible peptide linker LL(RADAAAA(G4S)4)using overlapping PCR.At the same time,a signal sequence of expression vector p PIC9 K was added at their N-terminus and a 6-His TAG was add at their C-terminus.The amplified products sc Fv and Bs Ab were cloned into expression vector p PIC9 K,and the recombinant p PIC9 K were digested with Sal ? and electro-transformated into Pichia pastoris.The clones selected by G418 was picked and induced by methanol.Through analysis by SDS-PAGE and Western blot,these recombinant antibodies were expressed in this system as secreted proteins.In summary,we acquired anti-merozoite surface protein of malaria sc Fv and anti-outer membrane protein of Orientia tsutsugamushi /anti-red blood cell membrane nonagglutination protein Bs Ab gene fragments by overlapping PCR.The sc Fv was cloned into expression vector p PIC9 K and expressed successfully in this system through analysing by SDS-PAGE and Western blot.The IFA result showed that the sc Fv had positive reaction with plasmodium merozoite surface antigen.The anti-outer membrane protein of Orientia tsutsugamushi/anti-red blood cell membrane nonagglutination protein Bs Ab was cloned into expression vector p PIC9 K and expressed successfully in this system through analysing by SDS-PAGE and Western blot.The red blood cell aggregation result showed that the Bs Ab had positive reaction with red blood cell membrane nonagglutination protein.Which had laid the foundation for its further applying for fast and accurate diagnosis of infectious pathogens such as Orientia tsutsugamushi in the early stage and Species identification.