Baculovirus Recombinant 北制Protein Expressio車師n Vectors (Dual Promoters)

The baculovirus vector是鄉 system is widely used for th如煙e expression of rec能場ombinant proteins in culture見就d insect cells. It is舞們 one of the most versatile and po呢物werful systems for eukaryotic妹得 expression of reco一如mbinant proteins. T慢媽his system is particularly adva男爸ntageous for large-scale preparat中歌ion of proteins that require expression很短 in eukaryotic host cells業服. Many eukaryotic proteins un頻林dergo posttranslational 技快modifications that can only tak玩業e place in eukaryotic cell體就s (e.g. glycosylation), or they 工司need a eukaryoti雨鄉c cellular milieu for proper foldin去弟g (e.g. membrane proteins). In門報 these cases, prokaryotic expression sy國舊stems are often inadequate and the b玩秒aculovirus expression system co他樹uld be a good alternative自遠.

Baculovirus is a double-stranded DN女高A virus that commonly infect玩森s insects, particula笑事rly members of t問內he order Lepidoptera (moths, butterfl劇劇ies and skippers). The cloning紅船 vector in our baculovirus expression 錯裡system, pBV, is optimized從兵 for use with the baculovirus shutt愛劇le vector (known as bacmid) derive爸體d from the baculoviru光西s strain AcMNPV (Autographa calif跳還ornica multicap地來sid nucleopolyhedrov睡歌irus), which has a 134 kb geno化少me in its wildtype form.市地

The gene of inte技可rest is first cloned into t著行he pBV vector under the control of身慢 a strong promoter. The pBV vector 很玩with dual promoters contains both 請相the P10 and poly購男hedrin (PH) promoters行術, derived from AcMNPV allowing users科短 to simultaneously expr和老ess two different genes of interes房鄉t. This makes the vector part器靜icularly suitable for application紅他s requiring the co-expression of公畫 two different g報答enes in cells such a新厭s studies involving the朋到 formation of protein complex媽車es or for following cellular enzymati音些c reactions. Since both P10 信照and PH promoters are derived from 行司AcMNPV, in order to reduce po報林ssibilities of recombin我跳ation between duplicated AcMNP資話V sequences, the P10 promoter is plac身暗ed in a reverse orientation relati笑笑ve to the PH promoter wit街頻hin the pBV vector.

The entire expression cassette consisti體會ng of the two promot聽玩ers and their downstream genes o算女f interest, along wi路我th a gentamicin resistance gene, is f快山lanked by the Tn7 transpos做市on terminal elements, Tn7L and Tn7R. 船東This vector is then transformed 去笑into E. coli carrying the bacmid雪唱 shuttle vector a船暗nd a helper plasm們開id. The bacmid is新子 essentially a very large plasmid con購外taining the bacu輛白lovirus genome modi兒厭fied to carry a lacZ gene and a你商n attTn7 docking site in湖笑serted in the la國明cZ coding region. The helper plasmi聽的d expresses the Tn7 transpo們影sase. The transposase w裡身ould then mediate the tran遠鐘sposition of the region flanke房區d by Tn7R and Tn7L on th樂西e pBV vector, whi對什ch contains the expression cassette 慢老for the two gen制會es of interest and gentamici很鄉n resistance, into the attTn7 docki志國ng site of the ba為到cmid. Colonies con身文taining recombinan河吧t bacmids can be identified by化河 gentamicin select鐘靜ion and blue/white screening (non-recom作醫binant colonies are blue due to lacZ ex吧窗pression whereas r唱好ecombinant colonies ar年呢e white due to disruption光都 of lacZ by transpo匠知son insertion). Purif木麗ied bacmid DNA can then be u務鄉sed to transfect insect cells to genera吃雨te live baculovirus, whi土房ch can be used to produce the recomb錯坐inant proteins o老花f interest.

The most commonly used cell line fo著對r expressing recombinant proteins from 司要baculovirus vectors is Sf9. This clo村公nal line was derived from ovarian北業 tissue of Spodoptera fru著機giperda (fall armyworm). This cell l妹在ine is adaptable to a var公窗iety of culture and media conditions, i著兒ncluding suspension or monolater 見窗culture and serum-free me路內dia. Larvae and other Le行議pidoptera cell li放暗nes have also been used e們問xtensively, and there ar身鐵e some reports of b相音aculovirus being an e都關ffective vector for照呢 mammalian cells.

For further inform車要ation about this vector system, plea做坐se refer to the pape近下rs below.