Contruction Of Recombinant Antibody Library

[PRACTICAL 1]

MTEB 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 electrophoresis.

amplified

DNA

product

quality

via

agarose

gel

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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[PRACTICAL 1] 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

VH

2 ul

TOTAL

VL

2 ul

Ligase

1 ul

Vector

1 ul

1 ul 10 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) 3|Page

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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 4|Page

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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 ‘heatshock’ 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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