Phage display

Question 1

Phage display

Answer 1

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

Question 2

fused proteins (antibody fragments)

Answer 2

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

Question 3

DNA encoding the antibody (fusion)

Answer 3

is found within the virion

Question 4

what sort of phage is used

Answer 4

a filamentous phage structyre

Question 5

name an example of a filamentous phage

Answer 5

M13 phage

Question 6

how long in length

Answer 6

200nm

Question 7

what sort of genome

Answer 7

ssDNA

Question 8

target antibody to be expressed

Answer 8

on the outside of the vision

Question 9

process of making a phage display library

Answer 9

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

Question 10

scFv =

Answer 10

Vl and Vh of an immunogoblin linked by a peptide spacer

Question 11

what allow expression of scFv on the surface of phages

Answer 11

fusion proteins between scFV and PIII minor coat proteins allow expression

Question 12

where are PIII minor coat proteins found

Answer 12

on the end of the phage

Question 13

Fab fragments of antibodies can be expressed on the

Answer 13

surface of a phage as fusion proteins with PIII

Question 14

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

Answer 14

pVIII

Question 15

where is pVIII expressed

Answer 15

along the length of the phage particle

Question 16

pVIII are useful for

Answer 16

the isolation of low affinity antibodies

Question 17

phage selection

Answer 17

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

Question 18

various different places where antibodies can be expressed on a phage

Answer 18

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

Question 19

end goal of phage display

Answer 19

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

Question 20

isolating phages

Answer 20

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

Question 21

phage selection

Answer 21

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

Question 22

phage display process overview

Answer 22

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

Question 23

to target particular disease molecules

Answer 23

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

Question 24

once a phage displaying the desired antibody has be identified

Answer 24

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

Question 25

e.coli used to

Answer 25

bulk up population of phage- lyse bacteria