pINFUSE-hIgG1-Fc1 vector高效IgG Fc融合蛋白大量表达载体-BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心

pINFUSE-hIgG1-Fc1 vector

Plasmid designed for the construction of Fc-Fusion proteins

-BioVector NTCC质粒载体菌种细胞蛋白抗体基因保藏中心

Product information

Map图谱：

ProduCt information

Co ntent:

- 20 µg of pinfuse-hig G1 -fc1 plasmid provided as

lyophilized DNA.

- 4 pouches of E. coli Fast-Media® Zeo (2 TB and 2 Agar)

sto rag e and stabi l ity :

- Product is shipped at room temperature.

- Lyophilized DNA should be stored at -20˚C and is stable 3

months.

- Resuspended DNA should be stored at -20˚C and is stable up to

1 year.

- Store E. coli Fast-Media® Zeo at room temperature. Fast-Media®

pouches are stable 18 months when stored properly.

Qual ity co ntro l:

- Plasmid construct has been confirmed by restriction analysis

and sequencing.

- Plasmid DNAwas purified by ion exchange chromatography and

lyophilized.

General ProduCt use

pINFUSE-Fc is a family of plasmid developed to facilitate the

construction of Fc-fusion proteins by fusing the effector region

of a protein to the Fc region of an immunoglobulin G (IgG).

pINFUSE-Fc plasmids yield high levels of Fc-fusion proteins.

The level of expression is usually in the µg/mL range. They can

be transfected in a variety of mammalian cells, including

myeloma cell lines, CHO cells, monkey COS cells and human

embryonic kidney (HEK)293 cells, cells that are commonly used

in protein purification systems.

pINFUSE-Fc plasmids allow the secretion of Fc-Fusion proteins.

As Fc-Fusion proteins are secreted, they can be easily detected in

the supernatant of pINFUSE-Fc-transfected cells by SDS-PAGE.

Furthermore, functional domains can be identified by

immunoblotting and ligand blotting.

Fc-Fusion proteins can be easily purified by single-step protein

A or protein G affinity chromatography.

InvivoGen provides pINFUSE-Fc vectors featuring Fc regions

containing introns from different species and isotypes. In

humans, there are four isotypes: IgG1, IgG2, IgG3 and IgG4. The

Fc region mediates effector functions, such as antibodydependent

cellular cytotoxicity (ADCC) and complementdependent

cytotoxicity (CDC). IgG isoforms exert different

levels of effector functions increasing in the order of

IgG4

Plasmid features

• human g enomi c igG1 -fc (with introns): The Fc region

comprises the CH2 and CH3 domains of the IgG heavy chain and

the hinge region. The hinge serves as a flexible spacer between the

two parts of the Fc-fusion protein, allowing each part of the

molecule to function independently. A short intron is present

between each region (one intron between the hinge and CH2 and

one intron between CH2 and CH3). The presence of introns is

known to enhance the level of gene expression as splicing is

known to promote rapid and efficient mRNA export

1

.

Human IgG1 dispays high ADCC andCDC, and is the most suitable

for therapeutic use against pathogens and cancer cells.

• hef1 -HtlV prom is a composite promoter comprising the

Elongation Factor-1α (EF-1α) core promoter

2 and the R segment

and part of the U5 sequence (R-U5’) of the Human T-Cell Leukemia

Virus (HTLV) Type 1 Long Terminal Repeat

3

. The EF-1α promoter

exhibits a strong activity and yields long lasting expression of a

transgene in v ivo. The R-U5’ has been coupled to the EF-1α core

promoter to enhance stability of RNA.

• mCs: The multiple cloning site contains several restriction

sites that are compatible with many other enzymes, thus

facilitating cloning.

• sV4 0 pan: the Simian Virus 40 late polyadenylation signal

enables efficient cleavage and polyadenylation reactions resulting

in high levels of steady-state mRNA4

.

• o ri: a minimal E. coli origin of replication to limit vector size,

but with the same activity as the longer Ori.

• CmV enh / hferl prom: This composite promoter combines

the human cytomegalovirus immediate-early gene 1 enhancer and

the core promoter of the human ferritin light chain gene. This

ubiquitous promoter drives the expression of the Zeocin™-

resistance gene in mammalian cells.

• em2KC is a bacterial promoter that enables the constitutive

expression of the antibiotic resistance gene in E. coli. EM2KC is

located within an intron and is spliced out in mammalian cells.

• Zeo : Resistance to Zeocin™ is conferred by the Sh ble gene from

Streptoalloteichus hindustanus The same resistance gene confers

selection in both mammalian cells and E. coli.

• βGl o pan: The human beta-globin 3’UTR and polyadenylation

sequence allows efficient arrest of the transgene transcription5

.

Methods :

Plasmid resuspension:Quickly spin the tube containing the lyophilized plasmid to pelletthe DNA. To obtain a plasmid solution at 1 µg/µl, resuspend theDNAin 20 µl of sterile H2O. Store resuspended plasmid at -20°C.sel ecti on o f bacteri a with E. co li fast-medi a®Fast-Media® is a fast and conv eni ent way to prepare liquid andsolid media for bacterial culture by using only a microwave. FastMedia®is a TB (liquid) or LB (solid) based medium that alreadycontains the antibiotic. Fast-Media® Zeo is available separately:#fas-zn-l (liquid), #fas-zn-s (agar).1- Pour the contents of a Fast-Media® pouch into a cleanborosilicate glass bottle or flask.2- Add 200 ml of distilled water to the flask3- Heat in a microwave on MEDIUM power setting (about400Watts), until bubbles start appearing (approximately 3minutes). do no t heat a cl o s ed co ntai ner. do no tauto cl av e fast-medi a®.4- Swirl gently to mix the preparation. Be careful, the bo ttl eand medi a are ho t, us e heatpro o f pads o r g l o v es and carewhen handling .5- Reheat the media for 30 seconds and gently swirl again. Repeatas necessary to completely dissolve the powder into solution. Butbe careful to avoid overboiling and volume loss.6- Let agar medium cool to 45˚C before pouring plates. Let liquidmedia cool to 37˚C before seeding bacteria.Note: Do not reheat solidified Fast-Media® as the antibiotic will bepermanently destroy ed by the procedure.references:1. Nott A, et al. 2003. A quantitative analysis of intron effects on mammalian geneexpression. RNA. 9(5):607-17.2. Kim DW et al. 1990. Use of the human elongation factor 1 alpha promoter as a versatileand efficient expression system. 91(2):217-23.3. Takebe Y. et al. 1988. SR alpha promoter: an efficient and versatile mammalian cDNAexpression system composed of the simian virus 40 early promoter and the R-U5 segmentof human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 8(1):466-72.4. Carswell S. & Alwine JC. 1989. Efficiency of utilization of the simian virus 40 latepolyadenylation site: effects of upstream sequences. Mol Cell Biol. 9(10):4248-58.5. Yu J. & Russell JE. 2001. Structural and functional analysis of an mRNP complex thatmediates the high stability of human beta-globin mRNA. Mol Cell Biol. 21(17):5879-88.