Generation of Plasmids Expressing ORF1-4 of PCV2 as a Novel Research Focus




Alexandria Bradian

Introduction

Porcine circovirus (PCV) is a 1.7 kb, single-stranded, non-enveloped, DNA virus with an unsegmented circular genome. The strain of interest for this research is PCV2. The genome of PCV2 is predicted to contain as many as 11 open reading frames (ORFs). There are four major ORFs—ORF1, ORF2, ORF3, and ORF4. The first encodes two proteins involved in DNA replication, the second encodes a capsid protein, the third is predicted to encode an apoptosis-inducing gene, while the fourth is predicted to encode an apoptosis-suppressing gene (Ren, 2016).

PCV2 is currently considered one of the most critical viral pathogens of the United States pig population. It is found worldwide, although the origin is unknown. Infection of PCV2 leads to lymphoid depletion, histiocytic infiltration, and immunosuppression. It can influence several cellular processes such as signaling pathways, miRNA expression, and oxidative stress (Ren, 2016). PCV2 is the primary causative agent of several syndromes collectively known as porcine circovirus-associated disease (PCVAD) (Gillespie, 2009). Currently, vaccines against PCV2 are widely used in commercial farms; however, since the vaccines do not induce sterilizing immunity, the virus keeps circulating even in farms applying vaccination (Kekarainen, 2015).

The major reason this virus should be of concern to researchers is because of its potential to become zoonotic. PCV2 infects and destroys nearly every tissue and organ within its natural host, infects and induces immunosuppression, and is capable of infecting and replicating in human cells in culture (Khayat, 2017). Because of this, further study of how PCV2 interacts with its cellular hosts is important for the development of therapeutics to intervene with PCV2 infection. The goal of this project was to isolate the four open reading frames associated with protein products and insert them into a pcDNA3.1D/V5-His-TOPO mammalian expression vector, with hopes of transfecting PK-15 cells with the plasmids, extracting protein products, and eventually creating antibodies to those proteins.

Materials and Methods

The first step of this experiment involved plasmid DNA purification. The PCV2 genome was given to the lab in a plasmid in DH5-alpha cells, so the genome first needed to be isolated.

Primers were developed for each of the four ORFs. Once primers were designed and ready, each ORF was amplified by polymerase chain reaction (PCR), using the PCR Cycling Parameters for PfuUltra II fusion Hs DNA polymerase protocol. The PCR products were then purified.

The PCR products were cloned into a TOPO mammalian expression vector: pcDNA3.1D/V5-His-TOPO. The ligated expression vectors were then transformed into TOP10 chemically competent E. coli, and the cells were plated. Colonies were selected and harvested in LB broth and ampicillin for over 24 hour.

The quick Mini-prep protocol from the lab was then conducted, and plasmids containing an insert were selected for. These plasmids were then purified and sent out for sequencing to GeneWiz.

The sequencing results for the plasmids containing ORF1, ORF2, ORF3, and ORF4 were BLASTed against the NCBI database. Each was compared to the Porcine circovirus type 2 complete genome.

Results

PCR results showed black bands of expected sizes for ORF1-4 on the agarose gel, meaning the primers designed for ORF1-ORF4 were all successful (Fig. 1).

It was found that ORF1 matched the NCBI database and showed 99% of identities (Fig. 2). ORF2 matched the databased and showed 99% of identities (Fig. 3). ORF3 matched the database and showed 99% of identities (Fig. 4a). ORF4 matched the database and showed 98% of identities (Fig. 4b).

Discussion

The main goal of this project was to isolate the four ORFs and insert them into the pcDNA3.1D/V5-His-TOPO mammalian vector, and this was done successfully.

As mentioned, the BLAST search showed that the ORF1-4 all matched the database with either 99% or 98% of identities. The significance of the very few base differences is unknown at this current time. Further analyzation and research will need to be done before any future steps are taken.

The end goal of this project was and still is to further study the ORF’s protein products, as they are recognized as the functional proteins of the virus, as well as eventually produce antibodies using the purified PCV2 proteins of interest. Because antibodies against this virus are not commercially available, that last step is of utter importance. Mechanisms of PCV2 cell cognition, attachment, and entry are still currently being researched and not very understood. We are hoping that this research may actually help prevent PCV2 infection in humans, if such a thing occurs.

Reference

Gillespie J, Opriessnig T, Meng X, Pelzer K, Buechner‐Maxwell V. 2009. Porcine circovirus type 2 and porcine circovirus‐associated disease. Journal of veterinary internal medicine 23:1151-1163.

Kekarainen T, Segalés J. 2015. Porcine circovirus 2 immunology and viral evolution. Porcine Health Management. 1:17.

Khayat R. "Mechanism of cellular recognition and entry by porcine circovirus 2.” City College of New York. 2017.

Ren L, Chen X, Ouyang H. 2016. Interactions of porcine circovirus 2 with its hosts. Virus genes 52:437-444.

Figures


Figure 1-PCR products for ORF1, ORF2, ORF3, and ORF4, along with negative controls, on a 1% agarose gel.


Figure 2-Aligned sequence for ORF1.


Figure 3-Aligned sequence for ORF2.


Figure 4-Aligned sequence for (a) ORF3 and (b) ORF4.


Abstract

Porcine circovirus (PCV) is a single-stranded, non-enveloped, DNA virus with an unsegmented circular genome. PCV type 2 (PCV2) infection is widespread and essentially all pig herds are infected with PCV2; however, few have PCV2-associated diseases (PCVAD). The virus is of great concern due to its impact on the farming industry and potential to become zoonotic. Because it is the common link among disease, it is vital to understand the biology of this virus for disease control. This project involved designing primers for the four major open reading frame (ORFs) of the viral DNA, amplifying the sequences of interest using PCR, cloning the ORFs into mammalian expression vectors, and sequencing the cloned material. In the future, this project aims to isolate the purified viral protein encoded by the ORFs and develop antibodies for laboratory use that are not commercially available.

Full Paper

Acknowledgments

I would like to thank Dr. Rubin for his help in designing this experiment, his guidance, and for making this project possible. I would also like to thank Anthony Evans and Catharina Grubaugh for all of their time, effort, and incredible guidance throughout this project.


This document was last modified 05/17/2017.
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