RESULT AND DISCUSSION SUMMARY CONCLUSION SCOPE OF THE STUDY - - PowerPoint PPT Presentation

“CLONING AND EXPRESSION OF HUMAN β DEFENSIN 2 IN Escherichia coli”

Submitted By

SEEMA ABRAHAM

• Reg. No: SJAHMGB006

2007-2009 Under the guidance of Dr. E. Sreekumar, Scientist C,Department of Molecular Virology, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram

INTRODUCTION OBJECTIVES MATERIALS METHODS RESULT AND DISCUSSION SUMMARY CONCLUSION SCOPE OF THE STUDY

 DEFENSINS:

• Defensins are effector molecules of the innate host defense system with

antimicrobial activity.

• The mammalian defensins can be subdivided into three main classes according

to their structural differences: the α-defensins, β -defensins and θ-defensins.

 HUMAN β DEFENSIN 2:

• Human beta-defensin-2 (hBD2), first discovered in 1997 in human skin.
• hBD2 is a cysteine rich cationic antimicrobial peptide with low molecular

weight.

• It is mainly produced by epithelial cells.

• DNA sequence of the pET-28a-hBD2, which codes for the target peptide

containing 49 amino acid with a molecular weight of 5.3 kDa. This includes the N (His) 6 Tag and the C-terminal 41 amino acid hBD2.

 MODE OF ACTION:

• HBD2 sequentially permeabilise the outer and inner membrane of E. coli.
• It form voltage dependent channels in membranes.
• It induce leakage of cytoplasmic content.

 FUNCTIONS:

• HBD2 kills bacteria and fungi on the surfaces of higher organisms.
• It also influence adaptive immunity by attracting immature dendritic cells.

APPLICATIONS:

• HBD-2 can be used in the treatment of chronic skin infections.
• It inhibits HIV replication in the intracellular environment.
• rec-hBD-2 can be used as an antigen for the generation of anti-hBD-2-

monoclonal antibodies.

STRAINS:

• E.coli JM109 is a convenient host for initial cloning of target DNA into

pET vector and for maintaining plasmids.

• E.coli BL21 DE3 pLys S strain is convenient for expression of HBD2.
• It carries the gene for T7 RNA polymerase under lacUV5 control.

• To clone the cDNA of Human β defensin-2 isolated from

human blood in bacterial expression system (pET 28a+).

• To express the cloned gene in E. coli BL21 (DE3) pLysS.

• Vectors
• pGEM -T Easy, pET28a (+).
• Host
• E.coli JM109, E.coli BL21 DE3 pLys S.
• Medium
• LB Broth, LB Agar.
• Antibiotics - Kanamycin(25µg/ml),Ampicillin(50µg/ml).
• Enzymes
• Taq DNA Polymerase, Nco I,Not I,T4 DNA ligase.
• Antibodies
• Monoclonal Anti-poly Histidine, mouse anti IgG HRP conjugate.
• Markers
• 100bp ladder,1kb ladder,protein marker, Prestained marker.

 Restriction digestion of pGEM-T-hBD2.

 Restriction Digestion of pET28a.  DNA Purification of hBD2 and pET28a by GFX Gel- Band Purification System.  Ligation of hbd2 and pET28 Transformation of E.coli JM109 with ligated pET - hBD2 Confirmation of Positive Clones by Colony PCR

Plasmid Isolation from Positive Clones by Alkaline Lysis Method.  Sequence Confirmation of pET-hBD2 clone Transformation of Expression host (BL21 DE3 pLysS) with pET hBD2 SDS- PAGE Analysis Cell Disruption and Solubilization of Expressed Protein Western Blotting

 Restriction Digestion of pGEM-T-hBD2

Restriction digestion of hBD2-pGEM-T Easy. Lane 1:Undigested pGEM -T Easy –hBD2. Lane 2:Digested pGEM-T Easy –hBD2. Lane 3:100bp Marker.

1 2 3

hBD2 fragment.

 Restriction digestion with the enzymes Nco I and Not I gave lower band

(hBD2 fragment) of size 160bp.The remaining upper bands were the backbone vector fragments.

 Restriction Digestion of pET28a

Restriction digestion of pET28a Lane 1: Undigested pET28a. Lane 2: Digested pET28a. Lane 3: 1kb Marker. 5 kb 5239 bp

 Digestion of pET28a with restriction enzymes NcoI and Not I gave a

single band of size of 5239 bp.

 DNA Purification of hBD2 and pET28a by GFX Gel-Band Purification System.

Purified hBD2 and pET28 a fragments. Lane 1: Purified hBD2 fragment. Lane 2: Purified pET28a fragment. pET28a fragment hBD2 fragment

 The single band in each lane shows the purified fragments of hBD2 and

pET28 a

 Ligation of Nco I / Not I –hBD2 and Nco I / Not I pET28

Overnight ligation was performed at 4°C and the ligated product was used for transformation.

 Transformation of E.coli JM109 with Ligated pET - hBD2

Transformed colonies.

 Confirmation of Positive Clones by Colony PCR

Confirmation of Positive Clones by Colony PCR Lane 1: Clone 1. Lane 2: Clone 2. Lane 3: Clone 3. Lane 4: Clone 4. Lane 5: 100bpMarker 200 bp 160bp

 Four colonies were isolated and confirmed its molecular weight as160 bp.

 Plasmid Isolation from Positive Clones by Alkaline Lysis Method.

Plasmid Isolation of positive colonies.

Lane 1: Plasmid.

 Sequence Confirmation of pET-hBD2 clone

Translated peptide sequence of the hBD2 –pET28 a clone and submitted sequence from the Genebank ( Accession NM_004942) which shows 100% similarity with HBD2 pET expressed peptide.

 Transformation of Expression host (BL21 DE3 pLysS) with pET hBD2

Transformed colonies.

 SDS- PAGE Analysis

Expression of hBD2 gene by induction with IPTG. Lane 1: BL21 DE3 pLys S, pET28a-hBD2 (0 hr Uninduced). Lane 2: BL21 DE3 pLys S, pET28a-hBD2 induction after 1 hour. Lane 3: BL21 DE3 pLys S, pET28a-hBD2 induction after 2 hour. Lane 4: BL21 DE3 pLys S, pET28a-hBD2 induction after 4 hour. Lane 5: BL21 DE3 pLys S, pET28a-hBD2 induction after 6 hour. Lane 6: Broad range protein marker. 6 kDa

 The induced band was visualized corresponding to the band below the

6 kDa protein of the polypeptide marker. 5.3kDa

5.3 kDa Localization of the expressed protein in the cell. Lane 1:BL21 DE3 pLys S Mock. Lane 2: BL21 DE3 pLys S, pET28a-hBD2 (Induced supernatant). Lane 3: BL21 DE3 pLys S, pET28a-hBD2 induction after 6 hr (Induced pellet). Lane 4: Broad Range Protein Marker (NEB).

 The protein was found to be in the insoluble inclusion body,

since the bands were found in the pellet loaded.

 Western Blotting

SDS-PAGE and Western blotting analysis of hBD2. Lane 1: Uninduced Mock. Lane 1: Uninduced Mock. Lane 2: Uninduced. Lane 2: Uninduced. Lane 3: Induced – Insoluble fraction. Lane 3: Induced -Insoluble fraction. Lane 4: Induced – Soluble fraction. Lane 4: Induced -Soluble fraction. Lane 5: Protein Marker. Lane 5: Prestained Marker. 6 kDa 5.3kDa

1 2 3 4 5

 The protein was released into the media after cell lysis and the

concentration was high in the supernatant.

• The restriction digestion of pGEM-T-hBD2 and pET28a was

done.

• The fragments were purified and ligated and transformed E.coli

JM109 were produced.

• The sequence confirmation was followed by the transformation
• f E.coli BL21 DE3 pLys S with pET-hBD2.
• The IPTG induction was performed and its results were

confirmed by SDS PAGE and Western blotting.

• This study confirmed that the cDNA of human β defensin 2

was successfully cloned in E.coli JM 109.

• Also confirmed that the fusion protein was expressed in

E.coli BL21 DE3 pLys S.

• The expressed protein was present in the inclusion bodies in

the bacterial cells .

• Recombinant-hBD-2 (rec-hBD-2) can be used as an antigen for

the generation of anti-hBD-2-monoclonal antibodies.

• It is used for immunohistochemical research of human tumour.
• Also used for the research of intracellular targets of hBD-2.
• HBD-2 can be used in the treatment of chronic skin infections.
• Human β defensin-2 can be used as an ideal therapeutic agent

avoiding the problems of acquired resistance.