Construction And Optimizing Of Gland-specific Expression Vector By Dairy Goat BLG Gene Regulatory Elements

Mammary gland bioreactor is made of milk protein gene promoter and regulatory region to guide exogenous gene expression in mammary gland. The quality of mammary gland bioreactor directly related to the level of exogenous genes. The key to mammary gland bioreactor is specific construction of expression vector, and promoter play an important role in construction of mammary gland expression vector. In order to realize the high and specific expression of foreign genes in transgenic animal mammary gland, this study using gene recombination technology, amplified the 5 ˊ-flank, intron 1, intron 2 and 3 ˊ UTR fromβ-lactoglobulin gene of saanen dairy goats, and constructed the eukaryotic expression vector,as the preliminary exploration of the promoter activity and expression of specificity.(1) We cloned the promoter of β-lactoglobulin gene by high fidelity polymerase chain reaction(PCR)(2.1 KB, including 5’ flanking region and part of the first exon), 3’UTR(the last exon, intron 6 and 3’flanking region) and the first and second introns. Sequencing analysis showed that the sequence conservative of 5’ flanking region of dairy goat BLG gene is superior to the 3’ end control components compared to other ruminants.(2) To sift the high efficiency of the promoter fragment, we amplified 13 segments from the promoter of BLG 2.1 kb, which are 2081 bp(-2041/+40), 1981 bp(-1941/+40), 1881 bp(-1841/+40), 1681 bp(-1641/+40), 1481 bp(-1441/+40), 1281 bp(-1241/+40), 1081 bp(-1041/+40), 881 bp(-841/+40), 681 bp(-641/+40), 481 bp(-441/+40), 431 bp(-391/+40),381 bp(-341/+40), 331 bp(-291/+40). Respectively inserted into the polyclonal loci of plasmid PGL4.10, then, transfected HC11, 293 T and goat fetal fibroblasts cells, respectively.We found that the promoter of BLG gene had tissue specificity and the activity of 431 bp(-391/+40) is higher than others’.(3) In order to study the role of introns in gene expression, we used pEGFP-C1 as the skeleton of the carrier, and removed its promoter CMV through the enzyme AseⅠ and NheⅠ, then replaced with the promoter of BLG 431 bp(-391/+40). And then used the intron 1,intron 2 and 3 ˊ UTR as regulatory elements to construct the vectors pE-BLGU11,pE-BLGIN1U11 and pE-BLGINU. Next, the results of quantitative PCR and Western blot experiment supported the conclusion that the introns of BLG gene can enhance the expression ofGFP on the level of mRNA transcription and protein translation, especially the first intron.In summary, this study cloned and analyzed the promoter sequences of β-lactoglobulin gene, and identified the optimal promoter sequence of BLG by gradient cut out. With vector pEGFP- C1, the role of introns of β-lactoglobulin gene in gene expression was preliminary confirmed. We succeed to construct the mammary gland specific expression vector using the regulatory elements cloning from the of goat β-lactoglobulin gene, followed by the further improving the expression level of exogenous gene and producing mammary gland bioreactor.