key: cord-0037611-melttpiq
authors: Yu, Tian-fei; Shao, Shu-li; Xu, Xing-jun; Lv, Jian-wei; Li, Ming
title: Express Transmissible Gastroenteritis Virus Spike Gene B and C Antigen Sites in Multiple Expression Systems
date: 2012
journal: Information Technology and Agricultural Engineering
DOI: 10.1007/978-3-642-27537-1_7
sha: 775ae7e062c5ad677bc42067803425417d4d2b2c
doc_id: 37611
cord_uid: melttpiq

In order to illuminate the antigenicity of porcine transmissible gastroenteritis virus (TGEV) spike protein B and C antigen sites, the truncated spike gene including B and C antigen sites of Chinese isolate TH-98 was expressed respectively in E.coli, baculovirus and pichia pastoris expression systems. Dot enzyme-linked immunosorbent assays (Dot-ELISA) based on these three recombinant proteins were developed preliminarily. Ten sera obtained correspondingly from ten piglets two months old which showed up clinical symptom were used for examination. The study indicates that the assays are rapid, reliable and sensitive and it has the potential for use as serological methods for TGEV diagnosis.

Transmissible gastroenteritis (TGE) is a highly contagious viral disease of swine characterized by vomiting, diarrhea, and dehydration. Its causative agent is transmissible gastroenteritis virus (TGEV), considered the principal etiologic agent responsible for dramatic outbreaks of diarrhea and high mortality of newborn pigs, in which mortality approaches 100% [1] [2] . Porcine respiratory coronavirus (PRCV) is believed to be a mutant of TGEV, as it has been shown to be genetically related to TGEV but has a selective tropism for respiratory tissue with very little to no replication in the intestinal tissue of infected swine [3] . TGEV and PRCV share high homology in genome and can generate the full cross-reaction neutralizing antibody [4] [5] [6] , so it is difficult to discriminate TGE in clinical diagnosis with traditional serological methods.

The truncated spike gene including B and C antigen sites which is absent in PRCV was expressed in E.coli, baculovirus and pichia pastoris in this research. Dot-ELISA assays based on these three recombinant proteins were developed to detect TGEV antibodies and could avoid antibody cross-reaction from PRCV theoretically.

TGEV strain named TH-98 was isolated from a suburb of Harbin, Heilongjiang province, P.R. China, and Swine testicle (ST) cell line was grown as monolayer in Dulbecco's modified Eagle medium (DMEM) (GIBCO, USA) containing 10% fetal calf serum (GIBCO, USA) and 5% CO 2 in air. Viruses were harvested by three cycles of freezing and thawing, cellular debris was removed by low speed centrifugation at 1.0×l0 4 g (HITACHI CR22E, Japan) at 4℃ for 25 min, and virions in supernatant were pelleted by centrifugation at 1.0×10 5 g at 4℃ for 1.5 h (HITACHI CR22E, Japan).

All the three pairs of primers were prepared according to the sequence of TGEV strain TH98 from a data base, GenBank (Accession no.AF494337). PGu/PGl contained EcoRⅠ or SalⅠ restriction enzyme site respectively, PHu/PHl contained PstⅠ or SalⅠ restriction enzyme site respectively, and PYu/PYl contained EcoRⅠ or NotⅠ restriction enzyme site respectively. All the primers contained artifical start codon or termination codon (Table. 1 ). 

RNA was extracted as described by Yin et al [7] , meanwhile the ST cell RNA was also extracted as the negative control. Extracted RNA 4μl was added into the below components: 5×reverse transcription buffer 1μl, dNTP mixture (2.5 mM) 4μl, RNase inhibitor 0.5μl, primer PYl 5μl, AMV reverse transcriptase 2μl (10 U), sterile water 3.5μl, gently mixed in an Eppendorf tube and incubated at room temperature for 10 min, then transferred to a water incubator at 42℃ for l h prior to stored at -20℃ until use in PCR. PCR amplification was performed using PE2400 PCR equipment (USA). PCR was in 100μl volumes, using 10μl of 10×buffer (50 mM KC1, 100 mM Tris-Cl pH8.3, 15 mM MgCl 2 , 0.1% gelate, 16μl dNTP mixture (10mM), 1μl cDNA, 5μl of primers and lμl ExTaq polymerase (0.5u Takara, Dalian, China). For the PCR the mixture was submitted to 30 cycles of amplification involving heating at 95℃ for 30s, 55℃ (E.coli expression), 53.5℃ (baculovirus expression), 56℃ (pichia pastoris expression) for 30s, and 72℃ for 60s, there was then a final extension time of 10min at 72℃. A 2μl aliquot of PCR product was visualized by agarose gel electrophoresis (2% agarose, 100 Vs for 20 min, 0.8μg/ ml ethidium bromide included in gel) and subsequent U.V transillumination. The purified PCR product was named ts, tg or ty respectively.

The amplified S gene (ts) DNA was digested with EcoRⅠ, SalⅠ and the resulting fragment was inserted into the EcoRⅠ, SalⅠ sites of the vector pGEX-6P-1 (Pharmacia Biotech, Inc., USA) to place the cDNA under the control of the tac promoter. The amplified S gene (tg) DNA was digested with PstⅠ, SalⅠ and the resulting fragment was inserted into the PstⅠ, SalⅠ sites of the transfer vector pBlueBacHis2A (Invitrogen Co., USA) to place the cDNA under the control of the polyhedrin promoter. The amplified S (ty) gene DNA was digested with EcoRⅠ, NotⅠ and the resulting fragment was inserted into the EcoRⅠ, NotⅠ sites of the transfer vector pPIC9K (Invitrogen Co., USA). The three recombinant vectors were sequenced by Sangon bio-company, Shanghai, China.

Expression of B and C antigen sites in E.coli and recombinant proteins purification was followed the procedures offered by the description of Glutathione S-transferase (GST) Gene Fusion System (amersham pharmacia biotech, third edition, revision 2). Expression of B and C antigen sites in baculovirus and pichia pastoris were followed Manual of Bac-N-Blue Transfection and Manual of Expression Guide and Methods for Expression of Recombinant Proteins in Pichia pastoris (Invitrogen Co., USA).

Twelve percent SDS polyacrylamide gel was used to analyze the three recombinant proteins. After electrophoresis, one of the gels was stained with Coomassie Brilliant Blue R250 to visualize the protein bands. The proteins of the other gel were transferred onto the NC membrane for the Western blot analysis. The diluted protein samples were spotted on the NC membrane for the Dot-ELISA. The membrane was blocked with 5% non-fat dried milk powder in PBS with 0.05%Tween20 (blocking solution). The membrane was probed with the TGEV immunized rabbit serum, in blocking solution (1:100). A peroxidase conjugated sheep anti-rabbit IgG (Promega, USA) was used as the secondary antibody (1:1000) and the signal was detected with H 2 O 2 and 4-Chlor-1-naphthol as a chromogenic substrate. Ⅰ illustrate). This was consistent with the expected molecular mass of the fusion protein of pGEX-6P-ts which consisted of the GST (26KD) and S gene B and C antigen sites subunit (14KD).

Two different MOIs, 5 and 10 were tested. Seed two 6-well plates with 10 6 cells in each well. The final volume in each well should be 1-1.5 ml. The harvest (post infection) was, 24 hours, 48 hours, 72 hours, and 96 hours. As shown in Fig. 1 . illustrate, recombinant baculovirus expressed 33 KD protein corresponded to the molecular weight of TG of TGEV at 24 hours post-infection and the protein was accumulated in high amount till 96 hours post-infection. No specific band was detected in the culture medium by Coomassie brilliant blue staining.

Sf9 cells were inoculated recombinant virus with MOIs, 5. Infected cells were harvested after 96h. The cell sediments were resuspended in PBS (PH7.4). After interruptible ultrasonic treatment, the lysate and supernatant were analyzed by SDS-PAGE. The result showed that recombinant protein was soluble bulk in supernatant (The result was not given).

The recombinant B and C antigen sites protein expressed into the yeast culture supernatant was identified on the bases of its molecular weight. Numerous bands were observed in the 14-116 KD molecular mass range. A sharp band was observed at molecular masses of approximately 40 KD by SDS-PAGE. The recombinant protein is the major protein component observed in the culture supernatant ( Fig. 1. Ⅳ illustrate). 

The antigenicity of the three recombinant proteins was analyzed by Dot-ELISA assay. When the amount of spotting is 750ng, the recombinant protein expressed in prokaryotic system shows the positive reaction in contrast with GST ( Fig. 2. Ⅰ illustrate).

When the amount of spotting is 100ng, the ultrasonic lysis supernatant of Sf9 cell infected by recombinant baculovirus show the positive reaction in contrast with ultrasonic lysis supernatant of Sf9 cell (Fig. 2. Ⅱ illustrate). When the amount of spotting is 50ng, the recombinant B and C antigen sites protein expressed into the yeast culture supernatant show the positive reaction in contrast with the GS115 cells transformed with pPIC9K plasmids (Fig. 2.   Ⅲ illustrate). 

. Analysis of the antigenicity of recombinant protein expressed in pichia pastoris expression system. Dots A 1 (100ng), A 2 (50ng), A 3 (25ng), A 4 (12.5ng) were the positive yeast culture supernatant; Dots B 1 (100ng), B 2 (50ng), B 3 (25ng), B 4 (12.5ng) were the GS115 cells supernatant transformed with pPIC9K plasmids.

Ten field sera obtained from ten piglets about two months old which showed typical signs of epizootic TGE used for examination and the ten corresponding sera were positive detected by Dot-ELISA (Fig. 3) . 

The spike protein of TGEV has been shown to contain four major antigenic sites (A, B, C, and D). Site A is the main inducer of neutralizing antibodies and has been previously subdivided into the three subsites Aa, Ab, and Ac. Site A contains the residues 538, 591, and 543, which are essential in the formation of subsites Aa, Ab, and Ac, respectively. The peptide 537-MKSGYGQPIA-547 represents, at least partially, subsite Ac which is highly conserved among coronaviruses. This site is relevant for diagnosis and could be of interest for protection. Other residues contribute to site B (residues 97 and 144), site C (residues 50 and 51), and site D (residue 385). Site C can be represented by the peptide 48-P-P/S-N-S-D/E-52 but is not exposed on the surface of native virus [8] . Site B is dependent on intracellular glycosylation and is complex and conformation-dependent. This site is formed by at least three epitopes. Although site B is conformation dependent, MAbs specific for this site can bind TGEV spike protein by immunoblotting providing that the samples were not treated with 2-mercaptoethanol. Most probably, renaturation of spike protein occurs during the blotting of the protein to nitrocellulose paper [8] . Site C is linear and continuous. It is recognized by MAbs in Western blot analysis after treatment of the virus with 2.5% SDS and 5% 2-mercaptoethanol; It is represented by synthetic nonapeptides derived from TGEV spike protein [9] ; It is present in recombinant products expressed in bacteria, which do not reconstitute the native spike protein [10] ; and it is formed in the absence of glycosylation [8] . In addition, because binding and sequencing studies indicate that site C is not present in the respiratory variants of TGEV, this peptide could be useful to discriminate serum from TGEV or PRCV infected animals. The purified recombinant protein expressed in prokaryotic system in this research, can be recognized specially by polyclonal antibody according to the research of Yang et al [10] .

Although, the three recombinant proteins shared the same antigen sites, the quantity of amino acids of the former exceed the latter (the recombinant protein expressed in eucaryotae has 227 amino acids, the recombinant protein expressed in prokaryosyte has 119 amino acids). If this difference has an effect on antigenicity between them or how deeply effect on antigenicity need more experiment to identify.

Recently, some new ways to detection TGEV were developed, such as real-time RT-PCR [11] [12] , but these ways need expensive equipment and reagent. It seems to unsuitable applied in open country, especially in the third world countries. So we developed the simple ways to detecting antibody induced by TGEV, and the results seem more reliable. The study indicates that the assay reported above is rapid, reliable and sensitive and it has the potential for use as serological method for TGEV diagnosis.

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