key: cord-017775-qohf9pxp authors: Loa, Chien Chang; Wu, Ching Ching; Lin, Tsang Long title: Recombinant Turkey Coronavirus Nucleocapsid Protein Expressed in Escherichia coli date: 2015-09-10 journal: Animal Coronaviruses DOI: 10.1007/978-1-4939-3414-0_4 sha: doc_id: 17775 cord_uid: qohf9pxp Expression and purification of turkey coronavirus (TCoV) nucleocapsid (N) protein from a prokaryotic expression system as histidine-tagged fusion protein are presented in this chapter. Expression of histidine-tagged fusion N protein with a molecular mass of 57 kDa is induced with isopropyl β-d-1-thiogalactopyranoside (IPTG). The expressed N protein inclusion body is extracted and purified by chromatography on nickel-agarose column to near homogeneity. The protein recovery can be 10 mg from 100 ml of bacterial culture. The purified N protein is a superior source of TCoV antigen for antibody-capture ELISA for detection of antibodies to TCoV. Turkey coronavirus (TCoV) is the cause of an acute and highly contagious enteric disease affecting turkeys of all ages. The disease is severe in 1-to 4-week-old turkey poults [ 1 ] . Turkey fl ocks that recover from natural or experimental coronaviral enteritis may develop lifelong immunity [ 2 ] . TCoV has been recognized as an important pathogen of young turkeys. TCoV infection causes signifi cant economic losses in the turkey industry due to poor feed conversion and uneven growth. Outbreaks of TCoV enteritis in turkey poults remain as a threat to the turkey industry. In order to rapidly diagnose and effectively control turkey coronaviral enteritis , development of an antibody-capture enzymelinked immunosorbent assay ( ELISA ) for detecting antibodies to TCoV is essential. Development of ELISA for detection of TCoV infection requires large amounts of TCoV antigen . Molecular cloning and expression of TCoV N protein were carried out for preparation of large quantities of highly purifi ed viral proteins. Coronavirus is an enveloped and positive-stranded RNA virus that possesses three major structural proteins including a predominant phosphorylated nucleocapsid (N) protein, peplomeric glycoprotein, and spike (S) protein that makes up the large surface projections of the virion, and membrane protein (M) [ 3 , 4 ] . The N protein is abundantly produced in coronavirus-infected cells and is highly immunogenic. The N protein binds to the viral genomic RNA and composes the structural feature of helical nucleocapsid. The N protein is a preferred choice for developing a groupspecifi c serologic assay because of highly conserved sequence and antigenicity. The nucleocapsid protein s of various RNA viruses, such as mumps, rabies, vesicular stomatitis, measles, Newcastle disease, and infectious bronchitis (IBV) viruses, have been used as the coating antigens in diagnostic ELISA [ 5 -10 ] . Prokaryotic expression is an economic and convenient system to prepare large amount of pure recombinant protein. In addition, the antigenic integrity of N protein expressed in prokaryotic system is expected to be maintained due to the lack of glycosylation. This chapter describes expression and purifi cation of TCoV N protein with a prokaryotic system for preparation of a large quantity of highly purifi ed viral protein, which can be used as coating antigen for antibody-capture ELISA for serologic diagnosis of TCoV infection [ 11 , 12 ] . 13. Add 2 μl of the above RT mixture to the PCR amplifi cation reaction (100 μl) with primers NF and NR. A mix of Taq and Pfu at 10:1 is recommended to maintain PCR fi delity (Table 1 ) . 2. The nuclease reagent Benzonase is added with 1 μl (25 units) for every ml of BugBuster reagent used. 4. Ampicillin antibiotic marker is on the expression vector pTriEx and chloramphenicol antibiotic marker is on plasmid pLacI in the expression host strain Tuner cells. Carbenicillin is recommended to be in place of ampicillin for better stability for pH changes throughout the bacterial cultures. 5. The binding capacity of 1.25 ml of His-Bind resin is 10 mg of target protein per column. As for any affi nity chromatography, the best purity of target protein is achieved when the amount of protein extract is near the binding capacity. 6. The purpose of 6 M urea is to improve resolution of the sticky inclusion bodies. The presence of 6 M urea does not affect binding of His-Bind resin to target N protein. 7. The suggested ratio of RNApure reagent to sample is 10:1. Excess amount of RNApure reagent has no negative impact. The lower ratio (5:1) in this step is intended to obtain higher concentration of viral RNA in the fi nal supernatants. If the upper aqueous phase after centrifugation at step 3 is more than half of the total volume, there is not enough RNApure reagent added. The appropriate reagent amount may be adjusted. Chloroform is applied at 150 μl for every milliliter of lysate. 8. The sample mixture with chloroform at this step can be stored at −70 °C or even lower temperature before proceeding to the next step. 9. Optional: Inverting the tube for 5-10 min for air-drying of RNA pellet is a helpful tip to completely remove any residual ethanol that may interfere the following RT reaction. 10. It is critical to make sure that the jellylike RNA pellet is completely dissolved into solution by repeat pipetting. 11. The synthesized cDNA in the RT reaction can be stored at −20 °C or even lower temperature until used. 12. PCR product may be purifi ed. The vector must be gel purifi ed due to the long digested fragment size above 30 bp. 13. The molar ratio between vector and insert is suggested at 1:2 to 1:5. The volumes in this step are illustrated for initial exploration. The concentration of digested vector and insert can be estimated by OD 260 or agarose gel electrophoresis with known amount of DNA of similar size in adjacent wells. The ligation reaction mixture can be stored at 4 °C until used for transformation or at −20 °C for longer term. 14. After plating, the leftover transformation mix can be stored at 4 °C for further plating in the following days at different amount if needed. 15. The starter culture can be prepared from a fresh colony on a plate or directly from a glycerol storage stock. An OD around 0.5 represents a culture at log phase when the cells are at the best condition to expand and for protein expression. 16. This usually takes about 2-3 h to reach the OD range. The higher the OD of starter culture in the previous step, the shorter the time to reach this OD range. 17. The centrifuge tubes should be weighed before and after collection of cell pellet for estimation of wet pellet amount and the volume of BugBuster to be applied in the next step. Frozen storage of cell pellets may improve the extraction effi ciency of BugBuster reagents through the freeze/thaw cycle. 18. It is important to completely resuspend the cell pellets for the best results of BugBuster extraction. Higher volume of BugBuster reagent does not have adverse effect. Roughly 10-20 ml of BugBuster reagent should be enough for cell pellets collected from a 100 ml of culture. BugBuster reagent can be added directly to frozen cell pellets. There is no need to wait for the temperature to return to room temperature. Protease inhibitors may be added at this step but usually not necessary. 19. It is critical but somewhat diffi cult to completely dissolve the sticky inclusion bodies. Repeat pipetting up and down until the protein solution is homogeneous. Any undissolved particles will clot the His-Bind column and affect the purifi cation process. It is advisable to centrifuge the dissolved inclusion body protein solution at 5000-10,000 × g for 10-15 min at 4 °C for clarifi cation before application to the column. 20. Eluate may be collected in fractions such as 0.5 or 1 ml each fraction. 21. The presence of 6 M urea is compatible with the protein assay reagent. The assay range can be adjusted for protein concentrations from low μg/ml to 1 mg/ml with different assay format. The protein concentration of the target N protein eluate as obtained following this process is about 1-2 mg/ml. The presence of 6 M urea has no adverse effect on plate coating for ELISA performance. Given the coating concentration of N protein at 20 μg/ml, the eluate is usually diluted in coating buffer for at least 1:10 to reduce the urea content to less than 600 mM and, subsequently, further diminish any possible effect on ELISA performance. Accordingly, the purifi ed N protein eluate can be directly applied to the ELISA assay for detection of antibodies to TCoV. Coronaviral enteritis of turkeys (blue comb disease) Immunity to transmissible coronaviral enteritis of turkeys (Blue comb) Identifi cation of the structural proteins of turkey enteric coronavirus Coronavirus immunogens Immunoglobulin class and immunoglobulin G subclass enzyme-linked immunosorbent assays compared with microneutralisation assay for sero-diagnosis of mumps infection and determination of immunity Rabies diagnostic reagents prepared from a rabies N gene recombinant expressed in baculovirus Baculovirus expression of the nucleocapsid gene of measles virus and utility of the recombinant protein in diagnostic enzyme immunoassays Immunological characterization of the VSV nucleocapsid (N) protein expressed by recombinant baculovirus in Spodoptera exigua larva: use in differential diagnosis between vaccinated and infected animals A diagnostic immunoassay for Newcastle disease virus based on the nucleocapsid protein expressed by a recombinant baculovirus Recombinant nucleocapsid protein is potentially an inexpensive, effective serodiagnostic reagent for infectious bronchitis virus Expression and purifi cation of turkey coronavirus nucleocapsid protein in Escheria coli Recombinant nucleocapsid proteinbased enzyme-linked immunosorbent assay for detection of antibody to turkey coronavirus The protocol "Recombinant turkey coronavirus nucleocapsid protein expressed in Escherichia coli " detailed in this chapter had been successfully carried out in the authors' studies on characterization and immunology of turkey coronaviral enteritis . Those studies were in part fi nancially supported by USDA, North Carolina Poultry Federation, and/or Indiana Department of Agriculture and technically assisted by Drs. Tom Brien and David Hermes, Mr. Tom Hooper, and Ms. Donna Schrader for clinical and diagnostic investigation, virus isolation and propagation, and animal experimentation.