Expression of recombinant proteins of porcine epidemic diarrhea virus using escherichia coli expression system

Name: Jiraporn Sritun

LCSH: Kasetsart University -- Dissertations. Ph.D. (Bio-Veterinary Sciences) 2021

Classification :.LCCS: QR201.V53

LCSH: Kasetsart University. -- Faculty of Veterinary Medicine -- Dissertations

LCSH: Viral diarrhea

LCSH: Escherichia coli

LCSH: Veterinary microbiology

LCSH: Swine -- Diseases

LCSH: Recombinant proteins

LCSH: Veterinary vaccines

Abstract: Porcine epidemic diarrhea virus (PEDV) is the cause of Porcine epidemic diarrhea (PED) which is the cause of serious watery diarrhea in suckling piglets. The spike (S) protein is one of the envelope protein and one of a major structural protein of PEDV. It plays an important role in the specific attachment with the host cell receptor and fusion of virus envelope with the host cell membrane. Both S1 and S2 domains of S protein containing neutralizing epitopes, therefore, it also plays important role in the host immune response. In this study, Thai isolates of PEDV were obtained from small intestines of infected piglets. S1 domain, S2 domain and overlapping part of S1 and S2 domains (S1-2) were amplified by reverse transcription polymerase chain reaction (RT-PCR) using specific primers for further recombinant proteins expression. The PCR products of S1, S2 domain and S1-2 segment were 2,127, 2,064 and 1,476 bps in size, respectively. The DNA sequences of these isolates were analyzed and has been shown to be G2 genogroup. In this study, the recombinant S1 domain, S2 domain and overlapping part between S1 and S2 domain (S1-2) proteins were expressed using Escherichia coli (E. coli). The purified PCR products of each segments were directly ligated with Directional pBAD202D-TOPO® vector and the mixture was used to transform Top 10 chemically competent E. coli. The optimum concentration of arabinose for the expression of recombinant either S1 or S2 or S1-2 proteins was 0.2%. The optimum time for the expression S1 and S1-2 protein was 8 hours whereas, S2 was 6 hours. The S1, S2 and S1-2 recombinant proteins were approximately 77, 75 and 54 kDa respectively. By Western blot analysis, S1, S2 and S1-2 recombinant proteins were specifically reacted with rabbit polyclonal antibodies against PEDV. The level of immunologic activity of each segment of recombinant S protein was different as determined by the minimal concentration of each purified recombinant proteins reacted with rabbit antibody against PEDV. The least amount of purified recombinant S1, S2 and S1-2 recombinant protein detected by 1: 100 rabbit polyclonal antibody was 1.6, 1.2 and 5 µg respectively. These recombinant S proteins may be a useful tool for the development of diagnostic test or subunit vaccine for PEDV.

Kasetsart University. Office of the University Library

Address: Bangkok

Email: tdckulib@ku.ac.th

Name: Theerapol Sirinarumitr

Role: Thesis Advisor

Name: Preeda Lertwatcharasarakul

Role: Thesis CO-Advisor

Created: 2021

Modified: 2025-07-11

Issued: 2025-07-11

วิทยานิพนธ์/Thesis

application/pdf

URL: https://www.lib.ku.ac.th/KUthesis/2564/jiraporn-sri-all.pdf

CallNumber: QR201.V53 .J57

eng

DegreeName: Doctor of Philosophy

Level: Doctoral Degraee

Descipline: Bio-Veterinary Science

Grantor: Kasetsart University

©copyrights Kasetsart University

ลำดับที่.	ชื่อแฟ้มข้อมูล	ขนาดแฟ้มข้อมูล	จำนวนเข้าถึง	วัน-เวลาเข้าถึงล่าสุด
1	jiraporn-sri-all.pdf	3.29 MB

ใช้เวลา

0.033123 วินาที