Cloning, Sequence Analysis Of CDS Of PRLR And Construction Of Adenovirus Vector For Rna Interference In Dairy Goat

Prolactin (PRL) plays a key role in growth, differential development, and functioning of mammary gland. It functions through binding with its specific receptor, prolactin receptor (PrlR). The PRL/PrlR signaling in mammary gland is an important part of milk biogenesis and secretion regulatory network. It has been recently discovered that PrlR-initiated signal pathways participate actively in milk lipid biogenesis and metabolism. There are several forms of PrlR, of which the long form PrlR (lPrlR) carries out most of the functions in mammary gland biosynthesis, while the short form (sPrlR) is believed to interact with lPrlR and thus interfere with its functioning. The present study cloned the coding regions (CDS) of long form PrlR from Xinong Saanen goat mammary gland tissue. The sequence was put to bioinformatic analysis and functional predictions to supply basic information of goat PrlR gene and its protein. Four pairs of short hairpin RNA oligonucleotides (shRNAs) based on the obtained sequence of goat lPrlR gene were designed, and one pair was selected and connected into adenovirus vectors to construct recombinant adenovirus vector used for RNA interference. Recombinant adenovirus vector was transfected into HEK 293 cells for its packaging and amplification, before further used in RNA interference experiments. The results:(1) Sequence of complete CDS of goat PrlR (long form) was obtained through RT-PCR and RACE PCR. The CDS of goat lPrlR (GenBank Accession No. JF966783) is 1746 bp in length, coding 581 amino acid residues. Similarity of nucleotide sequences between goat lPrlR and sheep, bovine, human and mouse lPrlRs was 97%, 95%, 81%, and 80%, respectively; similarity of amino acid sequences 94%, 91%, 67%, and 62%, respectively. Goat lPrlR has one transmembrane domain, locating within amino acid 237~258; Typical structures were located in the extracellular domain, and functional phosphorylation sites were observed in the intracellular domain of the protein sequence. The tertiary structure of goat lPrlR is an"L"shaped protein with the two"arms"connected by anα-helix.(2) Recombinant adenovirus carrying shRNA which targets goat lPrlR was successfully constructed, packaged and amplified. ShRNA were connected into shuttle vector pENTR/CMV-GFP/U6, which was then connected onto backbone vector pAd/PL-DEST via homologous recombination. The recombinant adenovirus vector was linearized with Pac I before used to transfect HEK 293 cells. After packaging and amplifying of recombinant adenovirus in HEK 293 cells, high-titered adenovirus was obtained (5.9×10~8 U/mL, LaSRT).In conclusion, the present study acquired the CDS of dairy goat lPrlR, and constructed recombinant adenovirus carrying shRNA designed for RNA interference study of PrlR goat in mammary gland.