Epitope Analysis and Immunological Studies of The Snake
Venom of Cobra Through Phage Display Technology
Advisor: Dr. Wen-Jen Yang
Institute of Biotechnology National University of Kaohsiung Student: Ping-Chieh Wang
Institute of Biotechnology National University of Kaohsiung
ABSTRACT
The way to deal with the snakebite patient is diagnosing rapidly who was bitten by what kind of poisonous snake and administrating the correct antivenin immediately. The preparation of antivenin was conducted by immunizing horses with detoxified snake venom, separating serum from collected horse blood, and purifying with ammonium sulfate. The titers of antisera during immunization period or final purified antivenins should be determined by enzyme linked immunosorbent assays (ELISAs) or venom neutralization tests. Both of these two methods need to use venom as antigen. However, poisonous snake is classified as preserve animal and it is difficult and dangerous to capture and breed snake and collect venom. Therefore, it is an important topic to develop a cheap, effective and easy-to-get venom substitute being used as antigen for relative assays. In this study, the phage display technology was used to search the crucial epitopes of snake venom and screen the epitope-carrying phages. It is expected that these phages can be used as venom substitute to resolve the snake venom hard-to-get problem and reduce the cost of assay.
Two phage displayed peptide libraries, ph.D.-7 and ph.D.c7c, were used to conduct the biopanning with antibodies of antivenin. Through the process of elution, amplification, and screening of three rounds biopanning, the number of high affinity phage population increases with the cycles of screening. The eluted phages number raised from 3.7x105 to 2.9x106 of the ph.D.-7 library, and 6x104 to 8x105 of the ph.D-c7c library, respectively. It indicates that the high affinity phage populations were selected through three times of biopanning.
Randomly select single phage from the third biopanning of ph.D.-7 and ph.D-c7c libraries, isolating phages DNA and sent to sequencing company for DNA sequence analysis. The results showed that the sequences of heptapeptides of SDPSSPS,
MNFGMAG, TLGFIPW, MNFGFMA, and ALFGLPP were found in 5, 4, 3, 2, and 2 times of ph.D.-7 library. Alignment with the heptapeptide sequences of the selected phage clones, four consensus sequence -D P S S P S, M N F G-M A-, --G/L F I/V P W and A/M L FG/P L P- were found. The heptapeptide sequence KSSLLRN selected from ph.D.-c7c showed that conserve with the “KSSLL”sequence of Taiwan cobra (Naja Naja Atra) venom (Accession No.1CHV_S). Also, the heptapeptide sequence
QADKHNK indicated that same “DKHN”sequence of Chinese cobra (Naja atra) cysteine-rich venom protein (Accession No. AY261468.1).
In the binding specificity test, the selected phages D23, D40, C2, C13, C14, C19, C29, C30, C31 and C33 can specifically bind to monovalent cobra antivenin IgG. The results show that these phages should carry the foreign amino acids corresponding to the epitopes of cobra snake venom. The results of dot blot indicated that these selected positive phages have the potential for serving as cobra venom substitute and developing diagnostic kit.
