Project 07/111 Final Report October 31, Project Title: Cloning and expression of porcine complement C3d for enhanced vaccines

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1 Project 07/111 Final Report October 31, Project Title: Cloning and expression of porcine complement C3d for enhanced vaccines Project Leader: Dr Douglas C. Hodgins ( Ex 54758, fax ) Department of Pathobiology Ontario Veterinary College University of Guelph Guelph, Ontario N1G 2W1 Co-investigator: Dr Patricia E. Shewen ( Ex 54759, fax ) Department of Pathobiology Ontario Veterinary College University of Guelph Guelph, Ontario N1G 2W1 Executive Summary Vaccines used in neonatal piglets have limited effectiveness because of immaturity of function of the immune system at this age and because of the immune suppressive effects of maternal antibodies absorbed by newborn piglets following ingestion of colostrum. Vaccines are needed which can induce active immune responses in piglets before maternal antibodies decline to nonprotective levels. Complement component C3d has been shown in laboratory studies in mice to have potent immune stimulating (adjuvant) properties. In addition, biochemical studies in lymphocytes suggest that C3d should block the suppressive effects of maternal antibodies, stimulating active immune responses. We have cloned the gene fragment coding for porcine complement C3d and have introduced modifications into the DNA sequence to permit expression of (recombinant) C3d in E. coli, for subsequent use in vaccines. Project Results Complementary DNA coding for a gene fragment of porcine complement protein C3 was generated using reverse transcription of messenger RNA extracted from porcine liver. A 1047 bp amplicon (coding for a gene fragment that included C3d) was produced using polymerase chain reaction (PCR) and gene specific primers based on published sequence (GenBank Accession # AF154933). The amplicon was ligated into the plasmid pcr 2.1-TOPO (Invitrogen) to facilitate sequencing and subsequent manipulation. The nucleotide sequence of porcine C3d was determined by the chaintermination method at the University of Guelph Molecular Supercentre. The sequence differed by three nucleotides from that published by a group in Germany (GenBank Accession # AF154933), resulting in two changes in amino acid sequence. One of these changes was in a region tentatively identified as the binding site for CD21, a receptor for C3d expressed on lymphocytes and may have functional implications. The sequence also differed by four nucleotides from partial sequence published by a group in China (GenBank Accession # DQ408206). These nucleotide changes code for two changes in amino acid sequence (Figure 1). It is anticipated that the use of recombinant

2 porcine C3d based on gene sequence derived from a pig with local York-Landrace breeding is preferable over using off-shore sequences, in producing vaccine constructs for the Ontario pork industry. A DNA fragment coding for: a) a biotinylation signal sequence b) a linker sequence c) a cysteine to serine mutation in the N terminal region of C3d d) Sca1 and BamH1 restriction enzyme cut sites was amplified by PCR (using a bovine C3d construct prepared in a previous project, as template), and ligated into the plasmid pentr/sd/d-topo (Invitrogen). A Sca1 cut site was engineered into the 5 region of the porcine C3d gene fragment, and a BamH1 site in the 3 region using PCR based mutagenesis. The porcine C3d amplicon was then ligated into the above mentioned plasmid construct. The resulting plasmid codes for porcine C3d with: a) a single site at the N terminus for biotinylation using BirA mediated enzymatic biotinylation b) a linker sequence to avoid steric hindrance between the biotin and the C3d protein in vaccine constructs c) a mutation of the N terminal cysteine residue, to disrupt the reactive thiol-ester bond present in native C3d Further study on the expression of the modified porcine C3d protein in E. coli will be undertaken. Milestones Initial RT-PCR amplification of porcine C3d was accomplished by February 28, The C3d gene fragment was cloned, and the sequence confirmed by July 10, The required mutations and alterations to porcine C3d to permit expression for use in vaccine constructs were completed by October 31, Project Expenditures A financial statement summarizing the project s financial status will be sent directly to Ontario Pork by the Financial Services department of the University of Guelph. Acknowledgement This research has been possible through the financial support of Ontario Pork and the Canada- Ontario Research and Development (CORD) Program, an initiative of the federal-provincialterritorial Agricultural Policy Framework designed to position Canada s agri-food sector as a world leader. The Agricultural Adaptation Council administers the CORD Program on behalf of the province.

3 Communications August 2007: The poster Cloning of porcine complement C3d as a potential vaccine adjuvant for neonatal pigs, was presented by summer student Sean Colyer at the Ontario Veterinary College, Summer Leadership Symposium (awarded first prize). October 2007: The cdna nucleotide sequence of porcine complement C3d has been submitted to GenBank (Bankit # ).

4 Figure 1: Swine C3d Nucleotide and Amino Acid Sequences CAC CTC ATC CAA ACC CCC TCC GGC TGT GGG GAG CAG AAC ATG ATC GGC ATG ACG CCC ACA His Leu Ile Gln Thr Pro Ser Gly Cys Gly Glu Gln Asn Met Ile Gly Met Thr Pro Thr GTC ATC GCT GTG CAC TAC CTG GAC AGC ACC GAA CAA TGG GAG AAG TTC GGC CTG GAG AAG Val Ile Ala Val His Tyr Leu Asp Ser Thr Glu Gln Trp Glu Lys Phe Gly Leu Glu Lys AGG CAG GAA GCC TTG GAG CTC ATC AAG AAG GGG TAC ACC CAG CAA CTG GCC TTC AGA CAA Arg Gln Glu Ala Leu Glu Leu Ile Lys Lys Gly Tyr Thr Gln Gln Leu Ala Phe Arg Gln AAG AAC TCA GCC TTT GCC GCC TTC CAG GAC CGG CTG TCC AGC ACC TGG CTG ACA GCC TAT Lys Asn Ser Ala Phe Ala Ala Phe Gln Asp Arg Leu Ser Ser Thr Trp Leu Thr Ala Tyr GTG GTC AAG GTC TTC GCT ATG GCA GCC AAC CTC ATC GCC ATC GAC TCC CAG GTC CTC TGT Val Val Lys Val Phe Ala Met Ala Ala Asn Leu Ile Ala Ile Asp Ser Gln Val Leu Cys GGG GCC GTC AAA TGG CTG ATC CTG GAG AAG CAG AAG CCT GAT GGA GTC TTC GAG GAG AAT Gly Ala Val Lys Trp Leu Ile Leu Glu Lys Gln Lys Pro Asp Gly Val Phe Glu Glu Asn GGG CCC GTG ATA CAC CAA GAA ATG ATT GGT GGC TTC AAG AAC ACT GAG GAG AAA GAC GTG Gly Pro Val Ile His Gln Glu Met Ile Gly Gly Phe Lys Asn Thr Glu Glu Lys Asp Val TCC CTG ACA GCC TTT GTT CTC ATC GCG CTG CAG GAG GCT AAA GAC ATC TGT GAA CCA CAG Ser Leu Thr Ala Phe Val Leu Ile Ala Leu Gln Glu Ala Lys Asp Ile Cys Glu Pro Gln GTC AAT AGC CTG TTG CGC AGC ATC AAT AAG GCA AGA GAC TTC CTC GCA GAC TAC TAC CTA Val Asn Ser Leu Leu Arg Ser Ile Asn Lys Ala Arg Asp Phe Leu Ala Asp Tyr Tyr Leu GAA TTA AAA AGA CCA TAT ACT GTG GCC ATT GCT GGT TAT GCC CTG GCT CTA TCT GAC AAG Glu Leu Lys Arg Pro Tyr Thr Val Ala Ile Ala Gly Tyr Ala Leu Ala Leu Ser Asp Lys CTG GAT GAG CCC TTC CTC AAC AAA CTT CTG AGC ACA GCC AAA GAA AGG AAC CGC TGG GAG Leu Asp Glu Pro Phe Leu Asp Lys Leu Leu Ser Thr Ala Lys Glu Arg Asn Arg Trp Glu GAA CCT GGC CAG AAG CTC TAC AAT GTG GAG GCC ACA TCC TAC GCC CTC TTG GCT CTG CTG Glu Pro Gly Gln Lys Leu Tyr Asn Val Glu Ala Thr Ser Tyr Ala Leu Leu Ala Leu Leu GTA GTC AAA GAC TTT GAC TCT GTC CCT CCT ATT GTG CGC TGG CTC AAT GAG CAG AGA TAC Val Val Lys Asp Phe Asp Ser Val Pro Pro Ile Val Arg Trp Leu Asn Glu Gln Arg Tyr

5 TAC GGA GGT GGC TAT GGA TCT ACC CAG GCC ACT TTC ATG GTG TTC CAA GCC TTG GCC CAA Tyr Gly Gly Gly Tyr Gly Ser Thr Gln Ala Thr Phe Met Val Phe Gln Ala Leu Ala Gln TAC CAG AAG GAT GTC CCT GAT CAC AAG GAT CTG AAC CTG GAT GTG TCC ATC CAC CTG CCC Tyr Gln Lys Asp Val Pro Asp His Lys Asp Leu Asn Leu Asp Val Ser Ile His Leu Pro AGC CGC AGC Ser Arg Ser Nucleotides highlighted in green differ from sequence derived in Germany (GenBank Accession #AF154933). Amino acids which are double underlined differ from amino acid sequence from the same source. Nucleotides highlighted in pink differ from sequence derived in China (GenBank Accession #DQ408206). Amino acids which are single underlined differ from amino acid sequence from the same source. Amino acids highlighted in yellow have been tentatively identified as a binding site for CD21, a receptor for C3d on lymphocytes (Lambris et al., 1985, Proc. Natl. Acad. Sci. USA, 82: ).