Cloning and Sequencing of Sialoadhesin and CD163 cDNA from Porcine Reproductive and Respiratory Syndrome virus infected Porcine Alveolar Macrophages

Abstract

Sialoadhesin (Sn) and CD163 receptors were reported as two essential receptors on porcine alveolar macrophages (PAM) for PRRSV infection and importance for the study of the immunity of pigs after infection. The objectives of this study were cloning and sequencing the porcine Sn (full and N-terminal domain) and CD163 (full and domain 5) cDNA from 3 strains of PRRSV infected PAM of Thai nursery pigs and comparison of gene expression level with GAPDH gene as internal reference. In this thesis, total RNA was extracted from 6 positive EU, US and HP-PRRSV infected PAM. The porcine Sn (full and N-terminal domain) and CD163 (full and domain 5) cDNA were further amplified using PCR technique. Band densitometry was used to quantify expression level. Then, the cDNA were cloned into pCR®-XL-TOPO® (full cDNA) and pCR®8-GW-TOPO® (N-terminal domain and domain 5 cDNA) vectors. The nucleotide and deduced amino acid sequences including compositions were then analyzed and compared. The results showed that 10 clones of Sn (full) and 20 clones of CD163 (full) were constructed in pCR®-XL-TOPO® vectors, while 24 clones of Sn (N-terminal domain) and 29 clones of CD163 (domain 5) were transferred into pCR8®-GW-TOPO® vectors. The sequences and compositions of nucleotide and deduced amino acids of these genes from 3 strains of PRRSV were successfully analyzed and showed more than 95% similarity. Furthermore, the US strain of PRRSV infected PAM collected from Thai nursery pigs showed higher expression level of porcine Sn and CD163 cDNA than that of the HP and EU strains.