Phage display Flashcards

1
Q

Phage display

A

a technique in which antibody fragments are expressed as a fusion coat protein on bacteriophage

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

fused proteins (antibody fragments)

A

are displayed on the surface of the vision, while the DNA encoding the fusion resided within the virion

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

DNA encoding the antibody (fusion)

A

is found within the virion

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

what sort of phage is used

A

a filamentous phage structyre

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

name an example of a filamentous phage

A

M13 phage

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

how long in length

A

200nm

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

what sort of genome

A

ssDNA

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

target antibody to be expressed

A

on the outside of the vision

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

process of making a phage display library

A

1) B cell source (immunised animal)
2) production of mRNA
3) production of cDNA from mRNA
4) PCR to amplify each the the V-gene facilities ( VH and VL)
5) clone as scFv onto PIII of phage

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

scFv =

A

Vl and Vh of an immunogoblin linked by a peptide spacer

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

what allow expression of scFv on the surface of phages

A

fusion proteins between scFV and PIII minor coat proteins allow expression

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

where are PIII minor coat proteins found

A

on the end of the phage

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Fab fragments of antibodies can be expressed on the

A

surface of a phage as fusion proteins with PIII

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

Fab fragments of antibodies can also be expressed on the surface of phages as fusion proteins with

A

pVIII

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

where is pVIII expressed

A

along the length of the phage particle

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

pVIII are useful for

A

the isolation of low affinity antibodies

17
Q

phage selection

A

1) phage antibody library
2) biopanning and washing
3) UV source and shadow stick selection
4) elution of bound phage antibodies
5) add to agar plate with e.coli and infection

18
Q

various different places where antibodies can be expressed on a phage

A
  • mainly expressed on the end (PIII) or along the length (PVIII)
19
Q

end goal of phage display

A

is to identify a phage producing an antibody with complementary VH and VL fragments of interest

20
Q

isolating phages

A

take population of phages- do ELISA and this allows you to pull out the phage producing the antibody of interest

21
Q

phage selection

A

need to eliminate all phages not producing antibody of interest

  • UV used to get rid of phages not of interest
  • phage of interest are protecting
  • these phages are then eluted and used to infect e.coli
22
Q

phage display process overview

A

1) take genes that encode a specific antibody and combine them on a strand of DNA
2) then the DNA is packaged into a protein coat made from a virus which only infects bacteria (bacteriophage)
3) when antibody gene are transcribed and antibody hat is formed not he top of the phage
4) phage antibody ‘hats’ are unique and bind to a specific disease target molecule 9change the antibody gene and you change the type of antibody hat)
5) many antibody phage with different hats are produced
6) library is a source of dining new antibody medicine

23
Q

to target particular disease molecules

A

the correct phage antibody has to be selected from the library
e.g. pathogen/antigen can be was he over the library to see which antibody complexes with it

24
Q

once a phage displaying the desired antibody has be identified

A

take phage and infect bacteria e.g. e.coli

25
Q

e.coli used to

A

bulk up population of phage- lyse bacteria