PiggyBac Antibody Light Chain Ge文關ne Expression Vector你好

Recombinant ant場讀ibodies have extensive appl亮司ications in the fields o新街f diagnostics and therapeutic m草商edicine. Current長習ly, there are about 18輛嗎0 monoclonal antibody products approved音跳 worldwide targeting specif城黑ic proteins, such 老熱as PD-L1, HER2, IL-17R, and VEGF. The d件快evelopment of genetic engineering 錢用has facilitated successful produ錢市ction of these ther這科apeutic antibodies,體我 which have ideal tumor penetr跳計ation behavior, short 腦林retention times, and reduce場鄉d immunogenicity. These therape南看utic recombinant antibodies a們作re mainly produce事兵d in mammalian cells in vitro (e.g., 請們CHO cells), sinc地妹e the post-transcript生就ional modification in this expressio用木n system is closest to the in vivo pro個多cesses in humans得金.

All antibodies contain heavy and lig友店ht chains, both of which c花山onsist of variabl唱男e (V) and constan錯是t (C) regions. In黃姐 mammals, antibodies a弟黑re divided into five isotypes人煙 (IgG, IgA, IgM用從, IgD and IgE) based on the he對畫avy chain C region sequence. A gi黃哥ven mammalian antibody isoty劇時pe either has la秒懂mbda (λ) or kap場服pa (κ) light chains which are e高離ncoded by the light chain C region為花. While there is no apparen鐘服t functional differen南不ce, the ratio of the t他務wo types of light chains varies from 對愛species to species. The light chain pl懂地ays a role in preventing self-re數我activity, and there is growing e輛聽vidence that it enhances receptor activ子些ation through homotypic interactio為從ns. In addition to this constant regio船可n, the V regions in heavy and下動 light chains contri有風bute to antigen recognition. Therefor南現e, by customizing sequences encodin我睡g the light chain 行技V region, customers may generate vec雨員tors to satisfy their personalized ne不你ed for antigen-binding 遠話specificities. In the vec慢作tor design process, the light 內西chain C region m身什ay either be selected from our算可 popular light cha河海in C region data工讀base (containin弟算g human κ and λ2 light chains, and mous懂都e κ, λ1, λ2 light chains) o吃訊r be pasted by customers using thei自兵r own sequence. Ad我醫ditionally, signal購理 peptides (e.g., IL-2 sig) can be 跳自added into the vector to increase secre歌店tion of the recombinant antibody.

VectorBuilder’s piggyBac an店長tibody light chain expression ve時書ctor system is a highly efficient t謝開ool for achieving non-現商viral, transposon-based de技訊livery of antibod嗎章y light chain expres師日sion cassettes into mammal長呢ian cells. This system contains two vec不新tors, both of which ar南兒e engineered as 雜拍E. coli plasmid視吧s. The first vector is ref習服erred to as the helper PBas信個e plasmid, encoding the tran城錯sposase. The second vector is ref討道erred to as the piggyBac trans些內poson plasmid which contains two term裡拿inal repeats (TRs) bracketin鐘服g the antibody light chain ex化森pression cassette t林會o be transposed. When the helper P為醫Base plasmid and the piggyBac transpos農水on plasmids are co-件司transfected into targ黑笑et cells, the transposase from場音 the helper plasmid will reco我件gnize the two TRs on t街討he transposon and inser音民t the flanked antibody light chain exp內說ression cassette in動民to the host genome. Inser說開tion typically oc很技curs at host chromosomal sites tha費讀t contain the TTAA sequence, 拍門which is duplicated金讀 on the two flanks of 錯雜the integrated fragment. PiggyB資你ac is a class II transposon, mean弟到ing that it moves in a c票醫ut-and-paste manner, hopping f暗吧rom place to pl爸費ace without leaving copies behind. 弟人(In contrast, class I transpo慢下sons move in copy-and-past個器e manner). Because the helper plasm很相id is only transiently transfected笑呢 into host cells, it will get l靜輛ost over time. With 秒兒the loss of the 件拍helper plasmid, the integration o會鐵f genes encoding the antibod煙飛y light chain becomes permanent in t林農he genome of host cells.

For further information公器 about this vector system, please少店 refer to the papers我中 below.

Our piggyBac an在志tibody light chain expression vectors校年 are designed to drive hi個白gh-level permanent antibody light來裡 chain expression in mamma但媽lian cells under a user-selected promo數科ter when used in conjunct場筆ion with the helper plasmid.

Technical simplicity: Delivering plasmid vectors into cells 紅女by conventional trans山校fection is technically s好女traight forward, and far easier服國 than virus-based vectors which requi作請re the packaging o慢商f live viruses.

Permanent integration o務議f vector DNA: The piggyBac antibody light chain expre就朋ssion vector facilitates long-term exp兵藍ression of antibody light chains in樹員 mammalian cells by allowing開吃 integration of the transp作信oson carrying the expres個數sion cassettes into the host genome.影飛

Reproducibility a個志nd scalability: The recombinant pro文去tein is harvested directly fr新線om transfected host房不 cells. Therefore, reprodu看場cibility of different錢要 batches can be easily achieved說畫. Moreover, the ampl微站ification of host cells allows for lar也事ge-scale antibo報作dy production.

Limited cell type range: The delivery of piggyBac vectors into都如 cells relies on transfection.公書 The efficiency of trans還答fection can vary great朋用ly from cell type to cell t輛林ype. Non-dividing cells ar媽刀e often more difficult to transf你那ect than dividing cells,林公 and primary cells ar務用e often harder t離小o transfect than imm高費ortalized cell 輛購lines. Some important cell t空姐ypes, such as neurons and pancreati外麗c β cells, are notoriously difficu答飛lt to transfect. Additionally, plasmid 票樂transfection is largely limited to 輛器in vitro applicat家綠ions and rarely used in vi木體vo. These issues limit the use上媽 of the piggyBac sys說新tem.

Optimal expressio金討n ratio of the heavy to lig做工ht chain difficult to achieve: All antibodies consist of heavy chains我刀 and light chains. 窗來For successful antibody product不鐵ion, a precise expression ratio of門為 the heavy to l關員ight chain is req玩林uired. This can 呢村only be achieved by 用離co-transfecting a second plasmid expre上月ssing the antibody heavy chain. Ac雪匠hieving optimal expression of both hea樂子vy chain and light chain may be拿東 difficult to control due to expre視美ssion driven by different promoters i姐件n two distinct ve好畫ctors.

5’ ITR: 5’ inverted ter美中minal repeat. When a DNA sequence is f唱她lanked by two ITRs, the piggyBac transp花一osase can recognize the我媽m and insert the flanked re都高gion including the two ITRs in動吃to the host genome.

Promoter: The promoter that dri務書ves your gene of interest is placed h資要ere.

Kozak: Kozak consensus s師電equence. It is placed in front of the習業 start codon of the ORF of interes就劇t because it is靜花 believed to facilitate translat個紅ion initiation in eukaryot友吧es.

IL2-sig: Signal peptide of Homo sapiens inter呢到leukin 2. It facilitates the secre房風tion of protein.

Light Chain Variable Region (VH): Light chain variabl影村e region for antigen recognition.

Light Chain Constant Regio紅老n (CH): Light chain constant regio視說n encoding isotype.

rBG pA: Rabbit beta-globin 暗動polyadenylation signal門見. It facilitates transcript秒得ional termination of the upstream l志國ight chain cons少近tant region cassette.

Marker: A drug selection去年 gene (such as neomycin resistance), a 朋秒visually detectab讀舞le gene (such as EGFP), or a dual-repor些場ter gene (such 還見as EGFP/Neo). T很畫his allows cells transduced紙腦 with the vector to be一校 selected and/or visu她算alized.

3’ ITR: 3’ inverted terminal repea術場t.

Ampicillin: Ampicillin resistance 刀答gene. It allows t花腦he plasmid to be maintained by amp拍通icillin selection in E. coli.

pUC ori: pUC origin of replication. Pl地知asmids carrying this ori好體gin exist in high copy numbers 訊區in E. coli.