MTEB

[PRACTICAL 1] 2404

CONTRUCTION OF RECOMBINANT ANTIBODY LIBRARY

Objectives:

1. To know and understand the antibody library principle and mechanism.

2. To learn how to amplified DNA (VH and VL) PCR method.

3. To check the amplified DNA product quality via agarose gel

electrophoresis.

4. To learn how to construct the recombinant scFV antibody library.

Materials :

Mouse spleen mRNA, PCR mixture, cDNA (20 ng), Vh primer mix, or VL primer
mix, taq-polymerase, MGCL2, dntPmix, 10 x buffer, H2O, 1% agarose gel,
electrophoresis case, trans illuminator, vector (E. Coli), ice, icebox, incubator,
eppendorf tube, LB broth, 100 bp ladder, ligase.

Introduction :

Variable fragment (FV) plays a role in antigen-binding activities of an

immunoglobulin molecule. It is the smallest unit of immunoglobulim, due to this
advantage, FV fragment is easier to manipulate than a whole antibody molecule
and it has become very useful for many applications.

Single-chain variable fragment (ScFv) refers to recombinant antibody

fragments consisting of only the VH and VL domains connected by a peptide
linker. ScFV’s can be constructed from various sources most commonly from
hybridoma or mouse immunoglobulin. Sheep immunoglobulin, chicken
immunoglobulin or chicken hybridoma and human antibody repertoire have also
been used templates. Today, ScFV can be relatively easily engineered using
recombinant DNA techniques in bacteria.

Methods:

A) VH and VL amplication from mouse spleen cDNA using thermal

cycler.

1. mRNA from the mose spleen were isolated

2. mRNA were converted to cDNA

3. Amplication of cDNA to form VH and VL were carried out using PCR

4. PCR mixture contained the following solution

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PCR mixture

cDNA (20 ng) 0.5 ul

VH primer mix 2.0 ul (for VL PCR, use VL primer mix)

Taq polymerase 0.5 ul

MgCl2 1.5 ul

dNTP mix 1.0 ul

10X buffer 2.5 ul

H2O 17.0 ul

TOTAL 25.0 ul

PCR condition

94’C 3 min (1 cycle)

94’C 1 min (30 cycle)

55’C 2 min (30 cycle)

72’C 2 min (30 cycle)

72’C 10 min ( 1 cycle)

B) Checking the amplified products (VH and VL) by electrophoresis

1. 1% agarose gel were prepared

2. Electrophoresis was carried out on PCR products

3. The results were observed under Trans illuminator

C) Construction of recombinant scFV antibody library

1. Ligation mixture was set up for VH, VL and vector

2. Ligation mixture was incubated at room temperature for about 5

minutes

3. Transformation mixture containing bacteria and construct were set up

4. The mixture was incubated on ice for 15 minutes

5. The bacteria was heat shocked at 42’C for 90 seconds

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6. Microfuge tube with bacteria is placed into the ice immediately

7. Microfuge tube is incubated on ice for 5 minutes

8. 1 ml of LB broth is pipetted into the microfuge tube

9. Incubation is carried out at 37’C with shaking for about 0.5 hour

10.8X serial dilution of 1:9 is done with the stock from centrifuge tube

11.Bacteria is placed onto a selctive plate

12.Plate is incubated at 37’C overnight and results are observed

Ligation mixture

H2O 3 ul Buffer 1 ul

VH 2 ul TOTAL 10 ul

VL 2 ul

Ligase 1 ul

Vector 1 ul

Results :

Parts A

The incubation is complete

Parts B

Based from the result obtained from transiluminator, the band size of VH
and VL by comparing to the 100 bp laddewr marker (GeneRuler), the size shown
at the ranges 300 bp to 2500 bp. The VH size band was slightly large than the VL.
On each well of agarose gel, the izes band of VH and VL are observed and this
proves that the VH and VL has been successfully amplified through PCR process.

Parts C

Agar plate with dilution Colonies

1 X 105 xx (9)

1 X 106 xx (0)

1 X 107 xx (0)

1 X 108 xx (0)

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Total antibody library that are successfully constructed :

= 9 X 105

Discussion :

The construction of recombinant antibody library is the storage mechanism

of a genetic information. For this experiment, we are using this technique to
construct recombinant antibody by using c DNA. cDNA is produced from mRNA by
using a reverse transcriptase mechanism and lacks introns. In other words, the
reverse transcription mechanism is used whereby genetic information contained
in mRNA is converted back into a double stranded DNA form. This means the VH
and VL from cDNA is amplified more effective.

VH primer mix is functions to attach the cDNA at specific initiation sites

and begin its amplication to form more VH strands. The function of the MgCl2 is
to stabilized the cDNA structure during anealing and elongation of the cDNA.
dNTP provides the substrates during elongation of new strands. While buffers is
functions to maintain the pH of the the mixture at suitable pH. Mainly to avoid
from enzymes denatured. The machine that we used for PCR in this experiment is
called the thermal cycler. The cDNA is assigned to 30 cycles of PCR amplified
using its own primers; either VL or VH primer mix. The 3 major steps in PCR
reaction :

Denaturation at 94’C :

The cDNA is melting which will opened up the DNA double stranded to a single
stranded band.

Anealing :

The primers will form an ionic bonds, template and is ready to copy the template.
Polymerase is presence highly in this stage.

Elongation at 72’C :

72’C is chosen as the polymerase is stable at this level of temperature. Other

bases that complement to the template are coupled from the 3’ side of template.

On part B explained the amplication of elctrophoresis using agarose gel to

ensure the amplication of VH and VL did occur successfully and are the same to
its template. To do this we compare the size of the band of the amplified DNA to
a 100 bp ladder from GeneRuler. The size of the VH is betweeen 300 – 500 bp
while the VL is 300 bp compared to the marker. Transilluminator is used to take
the agarose gel photograph, as its UV light will visualize the band fromed from it.

While Part C is important to maintained and propagate the cDNA. To

accomplish this, the cDNA is inserted to an appropriate plasmid. For this
experiment, E.Coli has been choosen. The E.coli is ‘heat-shock’ at 42’C for 90

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seconds which is to make the bacterial membrane more permeable to VH and VL

in the ligation mixture. To prevent the bacteria from recovering due to the ‘heat-
shock’ we placed it on Luria-Bertani broth (LB). This is also make the cells to
express the genes on the transformed plasmid, including the antibiotic resistance
gene. The variable region in Ig consist of hypevariable areas as well as constant
regions. VH and VL primers are designed from the constant regins. The bacteria
that is tranformed woth the plasmid will survive on the ampicillin agar platem
because of that the bacteria is resistant to ampicillin and with that the bacteria
are survived.

Conclusion :

The antibodies that is constructed successfully is 9 X 105 types of

antibodies. The average size of VH and VL were between 300 and 400 bp. The
amplified cDNA were successfully achieved using the PCR method and the quality
is the same as its original template.

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