| Single-domain heavy chain antibodies (VHHS) are the variable domain of the naturally occurring heavy chain antibodies (HCAbs) in camelidae (camels and llamas) and shark. The molecular weight of VHHs is only about 15 kDa,1/10 of conventional antibodies. VHHs can be easily expressed at mg quantities of the soluble, properly folded forms in bacterial systems. The beneficial biochemical properties, such as good stability in high temperature or in extremely pH conditions, make that VHHs are widly used for reseach purpose or diagnostic or therapeutic applications. It has potential for application in food control and analysis.Deoxynivalenol (DON), or vomitoxin, is a mycotoxin of the trichothecene group produced by Fusarium species. DON commonly contaminates cereals and cereal-based foods worldwide. The toxicity effects of DON to animals are inducing weight losses, reduction of food consumption, decreasing of nutration absorption rate and reducing the immune function. Most countries and regions have set up official maximum levels of DON in feedstuffs and foods.Detection methods based on immunogy are sensitive, fast and sutable for testing large numbers of samples. In present, polyclonal antibodies and monoclonal antibodies are most commonly used in immunogy detection methods for DON. However, generating polyclonal antibodies or monoclonal antibodies are time consuming, costly and laborious since it typically involves the repeated immunization of animals and needs high technical skills. Alternatively, antibody library devoid these disadvantages and provided a powerful way to generat antibody.Present work described construction of phage display library using non-immuned Lama pacos lymphscets as the source for cloning repertoire VHHs. The library was panned against an artificial antigen DON-MBSA which was synthesized by conjugate DON with MBSA. The main research results are as follows:1 Using non-immuned Lama pacos lymphscets as the source of VHHs coding sequence, two primery library namely SNAL and NAL were constructed by semi-nested PCR and nested PCR respectively. Both of them comprised by more than 1×107 independent clones. After helper phage rescue, two phage display library were generated respectively and the titre of both libray was up to 1013 CFU/mL. Colony PCR and restriction enzyme digestion analysis shows that the libraries have good diversity and can be subjected for panning.2 By three rounds of panning SNA-PDL against DON-MBSA, three types of anmino acid sequences with specific DON binding activities were obtained. They are DON-binder 1 (B9,B10,B11,B13,C3,C8,C10,C15), DON-binder 2 (C11), DON-binder 3 (A4,A9)。DON-binder 1 and DON-binder 2 have higher binding affility than DON-binder 3. By two rounds of panning NA-PDL against DON-MBSA, one type of clones encoding sigle-domain antibody was isolated.3 Recombinant phagemid pHEN-B9 was induced by IPTG in E.coli expression system. SDS-PAGE electrophsis of induced supernantant cell culture showed an espected band at about 50 kDa. Competitive ELISA showed the supernantant of induced cell culture can suppress DON monoclonal antibody binding with DON-MBSA, which indicated the peptide encoded by pHEN-B9 can be expressed with DON-binding activity.The creative points of this research are in that:1 Two non-immune phage display library (SNA-PDL and NA-PDL) were constructed by semi-nested PCR and nested PCR with primers optimized for Lama pacos, respectively. The libraries can be used as universe platform for panning different antigens.2 Three types of clones with specific DON binding activities were obtained by panning phage display library SNA-PDL. One clone with higher binding affinity was picked and expressed in E.coli expression system. The data showed that the peptide coded by the clone can be expressed with DON-binding activity, which indicated it has potential to be used for establishing immunoassay of DON. One type of clones encoding sigle-domain antibody was isolated from NA-PDL.
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