鸡β防御素1基因密码子优化及其在真核细胞中的表达

Codon Optimization and Its Expression of Chicken β-defensin-1 Gene in Eukaryocyte

鸡β防御素1在鸡防御系统中起重要作用,为实现其在真核细胞中的表达,本研究利用RT-PCR从狼山鸡法氏囊中扩增出鸡β防御素1成熟肽基因,同时参照酵母密码子的偏好性,设计合成Gal-1成熟肽片段新基因,并在其3'端添加6×His序列以检测鸡β防御素1的表达情况,分别构建分泌型表达载体pPIC9K-Gal-1和pPIC9K-Gal-1-a-His,线性化后电转化导入毕赤酵母菌株GS115,G418抗性筛选高拷贝转化子,阳性克隆用甲醇诱导,Tricine-SDS-PAGE分析和Western-blotting鉴定表达上清液。结果表明,从狼山鸡法氏囊中扩增出鸡β防御素1成熟肽基因,其序列与GenBank登录号AF033335的序列同源性为100%;Gal-1和Gal-1-a-His基因均成功整合到毕赤酵母基因组中,重组酵母蛋白Gal-1-His获得了成功表达。本研究为后续鸡β防御素1的高效表达和生物学活性研究奠定了基础。

Gal-1 plays an important role in defense system of chicken. To produce Gal-1 in eukaryotic cells, cDNA sequence of Gal-1 from the bursa tissue of Nantong Langshan chicken was cloned by RT- PCR. Then according to the requirement of yeast codon bias for protein expression, the new gene of Gal-1 mature fragment was synthesized. Especially 6 x His gene was fused in 3＇ end of the Gal-1 to fa- cilitate the detection of expression of Gal-1. The gene Gal-1 and new synthesized gene Gal-1-a were separately inserted into inducible secretion vector pPIC9K to yield the recombinant plasmid pPIC9K- Gal-1 and pPIC9K-Gal-1-a-His. The plasmid pPIC9K-Gal-1 and pPIC9K-Gal-1-a-His were linearized and transformed into P. pastoris GSll5 by electroporation. The positive recombinant Pichia strains screened with Geneticin were induced with methanol and the culture supernatant was identified by Tricine-SDS-PAGE and Western blot analysis. Results showed that the cloned cDNA of Gal-1 sequence had 100% homology with the sequence of GenBank accession number AF033335, the fragment of Gal- l and Gal-l-a-His were integrated into genome of P. paztoris GS115, and the His-fusion Gal-1 peptide was successfully expressed. This study would provide a solid basis for the studies on high expression and bioactivities of Gal-1.