Team:Beijing BHSF/Project

Our Project

Abstract

In this project, YFAL serves as a trans-locator to display high variable sections of A or B antibodies with regard to blood antigens on bacterial surface as single-chain variable fragment, ScFv. At the same time, green fluorescent proteins (GFP) and red fluorescent proteins (RFP) are presented in E.coli with A or B antibody. By incubating E.coli producing ScFv and GFP or RFP and cells containing blood antigen, scientists can analyze blood antigen of cells qualitatively and quantitatively if the corresponding antigen exists on target cells. We aim to utilize the bacterial surface display method in this experiment in order to develop a new technique to evaluate blood antigen of certain cells in human body.

Background

ABO antigens are presented on the surface of the Red Blood Cells. ABO antibodies circulating in the plasma of the blood can bind with corresponding ABO antigens, resulting in agglutination action. Antigens are proved to present on the surface of epithelial cells and endothelial cells with quite a high density. Moreover, antigens on the surface of cells are still active even within a certain time after cell death. Given the fact that some researches show that blood antigens on the surface of tumor cells from epithelial cells are abnormal, malignancy can be predicted by analyzing blood antigens on the surface of abnormal cells. These unique traits allow blood typing to provide essential functions in blood transfusion, criminal identification and even malignancy prediction.

General idea

The current blood typing approaches include antigen-antibody combination (slide agglutination or absorption-elution method) and genome sequence variance analysis (PCR-RFLP or PCR-sequencing based method). This project aims to determine blood type based on the antigen-antibody combination method. First, we gain basic information about ABO antigens and then artificially synthesize gene sequence of heavy chain and light chain in hypervariable region of corresponding antibodies. Then, we recombine gene sequence obtained from artificial synthesis with the gene sequence of YFAL on the surface of E.coli and display it on the surface of E.coli that are previously signaled by RFP or GFP. The modified E.coli can combine with red blood cells, epithelial cells and endothelial cells due to the interaction between antigens on target cells and antibodies on E.coli, hence resulting in specific adhesion patterns that can be detected by fluorescence microscope so that blood type and the amount of blood antigens can be clearly determined.

Parts

1. yfal signal peptide- scfv anti-A length 1.2kb
2. yfal signal peptide- scfv anti-B length 1.2kb
3. Arac/pBAD-yafal signal peptide- scfv anti-A- Linker-yafal-terminator length 3.5kb
4.Arac/pBAD-yafal signal peptide- scfv anti-B- Linker-yafal-terminator length 3.5kb

Results



Future works

We aim to continue on figuring out better sequence of antibody so that it will function in more circumstances and gain more specificity.

Acknowledgement

Sincerely thank for the encouragement and instruction from Professor Chen Guoqiang of Tsinghua University
Sincerely thank for the help and instruction about experimental strategies and reagent application from Mr.Li Teng
Sincerely thank for the reagent support from New England Biolabs

References

1. W Thomas, E Trintchina, M Forero,V. Vogel and EV. Sokurenko (2002) Bacterial Adhesion to Target Cells Enhanced by Shear Force. Cell, Vol. 109, 913–923,
2. HJ Ko, E Park, J Song, TH Yang, HJ Lee, KH Kim, and IG Choia (2012) Functional Cell Surface Display and Controlled Secretion of Diverse Agarolytic Enzymes by Escherichia coli with a Novel Ligation-Independent Cloning Vector Based on the Autotransporter YfaL. Applied and Environmental Microbiology Appl. Environ. Microbiol. 78(9):3051.
3. Nickerson KG Tao MH, Chen HT, Larrick J, Kabat EA (1995) Human and mouse monoclonal antibodies to blood group A substance, which are nearly identical immunochemically, use radically different primary sequences. J Biol Chem. 270(21):12457-65.
4. Wang CJ, Tang JQ, Li XF, Pan XZ and Cao M (2001) Expression and analysis of ScFv antibody of mcAb 50A anti-RBC blood group A substance. J Cell Mol. Immunol. 17(3):271-273