the development of vaccine against

(1)

'Construction of plasmids expressing specific genes of Mycobacterium tuberculos.is: towards

· tuberculosis'

Mustaffa Musa, Salwana Ahmad, Jamaruddin M. Asan, Zainul F.

Zainuddin,

Project number: 06-02-05-8012 IRPA 1999-2001

End Of Project Report

December 2001

'

¹

1\

(2)

•

-t

'

End Of Project report

Project number: 06-02-05-8012

Project Title:

'Construction of plasmids expressing specific genes of Mycobacterium tuberculosis:

towards the

development

of

vaccine against tuberculosis'

Project leader: Prof. Madya Dr. Mustaffa Musa

Tel: 7651700 ext. 2150 Fax: 09-7653370

Summary for the MPKSN Report

(For publication in

the

Annual MPKSN Report, please summarise the project objectives, significant results achieve, research approach and team structure)

The objective of the project was to oonstrud plasmid(s) expressing specific genes of Mycobacterium tuberculosis (MTB): towards the development of potential DNA vaccine candidates against tuberculosis. Several T -cell epitopes from various specific genes of MTB were selected which include ESAT-6, MTP40, MPT64 and · 38k0a. In order to construct the synthetic gene consists of those selected epitopes, the innovative PCR technology known as 'Assembly PCR' was employed. In this project, the synthetic gene (consist of multiple epitopes of M. tuberculosis) was successfully constructed and the gene is designated as vacll. The vacll was then cloned into plasmid DNA vector, pJW4303. The recombinant plasmid containing the vacll designated as pJWvacll was obtained. The DNA sequence of the cloned gene (vacll) or the insert was confinned by DNA sequencing method. So, in this project, we have constructed one DNA vaccine candidate for tuberculosis. Further studies that to be done to evaluate this vaccine candidate include immunogenecity and protection efficacy in animal model.

In this projed, the immunogenicity of pJWvacll was studied in mice in order to detennine its ability to induce various immune responses. Mice were immunised with the pJWvacll and control plasmid (pJW blank vector). At the end of the immunisation protocol, the spleenic lymhocytes were prepared from the two groups of mice.

Lymphocyte responses in vitro were determined

by Flow

Cytometric analyses, Lymphocyte Transformation Test and IFN gamma production {by ELISA) after culturing them with several peptides derived from the vacll construct.

Results of Flow Cytometry which measure lymphocyte activation status did not show any signifteant different between the control and test group. But, results of LTT and IFN gamma production showed that some mice vaccinated with pJWvacll were positive when compared to the control group. These results indicated that the DNA vaccine candidate (pJWvacll) is immunogenic in mice ie. able to induce immune response. The ability of pJWvacll to induce IFN gamma production suggested that the DNA vaccine construct stimulated the Th1 type of immune response which is essential for immunity to TB. However, more studies need to be done to confirm these prilimanary findings. Such studies include protection or challenge studies in mice but this part of the study can be done here due to lack of facility required.

2

UNIVERSITI SAINS MALAYSIA

DITERIMA

I .. ,2 DEC 2001 J

Bahaaian

R & D

p,,sat Penaajian Sain.s p

.. ··~~

I -;

(3)

0

'I

'

0

Objectives achievement •

• Original project objectives (Please state the specific project objectives as described in Section II of the Application Form)

-To clone genes/epitopes of M.tuberculosis into a plasmid (pCMV).

- To prepare DNA constructs/recombinant protein for vaccination.

- To study antibody and T -cell responses of the vaccinated animals.

• Objectives Achieved (Please state the extent to which the project objectives were achieved)

We have succesfully constructed a plasmid expressing multiple T -cell epitopes of M. tuberculosis as DNA vaccine candidate for tuberculosis.

• Objectives not achieved (Please identify the objectives that were not achieved and give reasons)

3

(4)

0

Technology Transfer/commercialisation Approach (Please describe the ap,proach planned to transferlcommerciaHse the results of the project)

This project has established recombinant DNA technology/approach especially assembly PCR technique for DNA vaccine development. This technology is not only usefull for TB but also for other diseases in future. This technology can be transferred to other research institutions/universities in this country.

Benefits of the Project (Please identify the actual benefits arising from the project as defined in Section Ill of the Application Fonn. For examples of outputs, organisational outcomes and sectoraVnational impacts~ please refer to Section Ill of the GuideHnes for the AppHcation of R & D Funding under IRPA)

• Outputs of the project and .potential beneficiaries (Please describe as specically as possible the outputs achieved and provide an assessment of their significance to users) ·

Laboratory set up/technology for DNA vaccines development

can

^be

a

trainning center for other reseachers in this country.

- The plasmid DNA construct (DNA vaccine candidate} obtained from this project has potential for further vaccine development as an alternative vaccine for the prevention and control of TB.

• Organisational Outcomes (Please describe as specifically as possible the organisational benefits arising from the project and provide as assessment of their significance)

estabfised training center for other reseachers/postgraduate students in this counby with regards to recombinant DNNPCR technology.

• National Impacts (If known at this point in time, please describe as specifically as possible the potential sectoral/national benefits arising from the project and provide an assessment of their significance)

Established linkage with Mycobacteriology Group, University of Sydney.

_ The clone product (DNA construct) obtained from this project has potential for further vaccine development as an alternative vaccine for the

prevention and control of TB.

4

(5)

0

..

Assessment of project structure

• Project Team (Please provide an assessment of how the project team perfonned and highlight any significant departure from plan in either structure or actual man- days utilised)

- Assoc. Prof. Dr. Mustaffa: overall management of the project, supervising immunogenicitiy studies (10 man-months)

Assoc. Prof. Dr. Zainul: organising PCR analyses and DNA cloning (4 man- months)

RO (Pn. Salwana Ahmad): performing all laboratory works (36 man-months)

• Collaborations (Please describe the nature of collaborations with other research organisations and/or industry)

CoJaboration with Mycobacteriology laboratory (Assoc. Prof. W. ·Britton). Centenary tnstitute for Cancer Medicine & Cell Biology, University of Sydney with regards to the infonnation on the latest technology in

DNA

vaccine development.

Assessment of Research Approach (Please highlight the main steps actually performed and indicate any major departure from the planned approach or any major difficulty

encountered) ·

Main steps:

1. Designing synthetic gene of M. tuberculosis consist of multipe T -cell epitopes of M. tuberculosis using a computer programme (Mac DNAsis & Oligo programme) and generation of oligonucleotides based of the gene construct (vacll).

Perfonning Assembly PCR to construct the vacll.

2. Cloning of the vacll gene into an expression vector (a DNA vaccine vector - pJW4303) and analyses of the recombinant clones by PCR, restriction digest and DNA sequencing.

3. Vaccination mice with pJWvacll and pJW4303 (control) for immunogenicity studies.

6. Lymphocyte Transformation Test and IFN gamma production assay.

Assessment of the Project Schedule (Please make any relevant comment regarding the actual duration of the project and highlight any significant va_riation from plan)

The project was running according to project schedule except at the beginning of the project due to time consumed for purchasing of reagents and training of the research officer.

5

(6)

NAMA PEN'fE\.lOIK fiiAM.A PROJEK

PENYA T.a.. PERBELI\NJAA.N BAG I TEMPOH BERAKHIR PADA.

PECAHAN KEPAI.A 8.A.KI TAl-l UN PERUNTUKAN BELANJA

- - - - {VOlt _ _ ~-f--J~.t:..u_

_

^_ ⁾⁰⁰⁰ ²⁰⁰⁰

11 OOQ G.'-JI t8.118.73 37,078.50 37,664.76

14000 EI.AUN 950.00 950.00 0.00

1500080NUS 558.50 1,871.50 0.00

:!1000 PERJALANAN 1.47~.00 7,600.00 1,046.00

~!2000 PENa"~NGKUTAfl. 0.00 0.00 Cr.OO

23000 PERHUBlNGAI~ 421.02 475.00 57.40

24000 SE\1\IAAN 0.00 ~800.00 0.00

25000 M.~N & MINUM 0.00 1,900.00 0.00

:!6000 BAHAN MENT AH 0.00 0.00 0.00

27000 BAH.A.N LAIN 15,148.74 26,600.00 21,196.21 28000 PEMBAJ KAN KEC 1,900.00 1.900.00 0.00 29000 HOSPI"rlliTY 413.75 475.00 282.45

36000 HART A MOOAL

o.oo

0.00 C•.OO

-··

JLIMI.AH BE~AFl 38,981.74 82,650.00 60,236.82

PINDAAN

UNIVERSITI

•

SAINS MALAYSIA KM\PUS ·cm~GAN KELAt-nAN

GeRAN PENYELIDIY-AN (R&D) JANGKA PANJANG

~AT PENGAJ\ANSA\NS PERUSATAN

~PSPISt 10243 ... ) OR MUSTAFFA MUSA

• •

CONSTRUCTION OF RECOMBINANT PLASf\UOS EXPRESSIPAG SPECIFIC OF MYCOBACTERIUM TUBERCUltlSIS TOW~RDS THE DEVELOPMENT OF VACONE TUBERCULOSIS.

30 NOVEMBER 2001

BAKI PERUNTUKAN JUML.Jd-1 BAYARAN PERSELANJAAN BAKI

PERUNTIJKAN

---~_.;, 200~-^f-- 200_1 _ _ PE~~ITU~-r-..-2001 _ _ ~~GGU~G.A~ 1-·-~tf!L

__

₁)<ESELURUI-IAN O.OC• 17,542.47 0.•30 17,542.47 35,135.10 0.00 35,1:!5.10 (17,692.63

0.00 1,900.00 0.•30 t,900.00 0.00 0.00

a.oo

1,900.00

0.00 2,430.00 0.00 2,430.00 1.397.80 0.00 1,397.00 1,032.20

0.00 8,027.00 0.00 8.027.00 0.00 0.00 0.00 8.027.00

O.OC• 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.00 8SS.62 0.00 838.62 9.00 0.00 9.00 829.82

O.OC• 3,8()1.00 0.00 3.800.00 0.00 0.00 0.00 3,800.00

0.00 1,9()1l.QD 0.1JO 1.900.00 0.00 0.00

o.ao

1,900.00

0.00 •J.OO O.•JO 0.00 0.00 0.00 0.00 0.00

0.00 20,651J.53 0.00 20.550.53 16,048.10 6.672.50 22,718.60 ('2,189.07:

0.00 3,80•].00 0.00 3.800.00 0.00 0.00 0.00 3,800.00

0.00 6015.30 0.00 606.30 205.50 0.00 205.60 400.80

0.00 0.00 0.00 0.00 0.00 0.00 0.(10 0.00

0.00 61,394.92 0.00 61,394.92 62,793.50 . 6,672.50 69,466.(10 1,928.92

-

-·M~ ·~·-··--

.. •-- -•• -·· •-"•• -- ·---.. -··-·-

'-~

----·

·---L--.-- - - -· --- .__ _________ - - - .. ---~

(7)

•

(J

Technology Transtertcommercialisation Approat:h ~lease desaibe the approach planned to transfer/commercialise the results of the project}

• Patent (P!esse state full title or the patent by gvmg the patent number or epp8cati<Jn number)

Nil

• Publication pertaining to.the research finding

. (a)

Report/Conference Paper

1. ·construction of synthetic genes of Mycobacterium tuberculosis using assembly PCR technique. 1oth MSMBB Scientific Meeting;14-16th May 2000, Hyatt Regency Saujana, Subang. (see Appendix B)

2. 'Construction of synthetic gene of Mycobacterium tuberculosis: towards the.

development of DNA vaccine against tuberculosis ... Global alliance for TB drug Development Conference on R & D coalition for TB drug development in Asia. May 2nd -4th, 2001. Penang Parkroyal Resort Batu Feringghi Beach, 11100 Batu Feringghi, Penang, Malaysia.

3.Norazmi Mohd Nor and Mustaffa Musa (2001 ). 'Potential approaches towards the development of a vaccine against tuberculosis: Recombinant BCG and DNA vaccine'. Monograph. Biotechnology Symphosia I. UMS.

(b) Journal Publication

(Use only the standard accepted abbreviations for journal titles. If there are none, give the fuD joumal title)

In preparation

(c) Others:

1. The Sun: 1oth June 1999 ... New

vaccine from

USM'.

2. Utusan Malaysia: 9th June 1999. '"USM researchers construct vaccine prevent

TB'

3. Segment 'Good Morning Malaysia', TV1 RTM 5/10/99 on 'TB vaccine research activities in PPSP'.

7

(8)

..

•

..

a Post Graduates (Who graduated or who are stiH participating the project)

Student Name

&Year of

^Thesis

Title

PhD/

Registration/Nation MSc

a

lily

Nil

• No. of Research Assistants or Officers funded by the project:

{a) Research Officers: 1 (b) Research Assistants: 1

Collaboration (Please describe the nature of collaborations With other research organizations and/or Industry)

Institutions:

(a) Local

Institutions:

Hospital

Kota

Bharu- provide blood

samples of TB

patients .

Year of completion

(b) International Institutions: Mycobacterial .Research Group,

Centenary Institute of Cancer Medicine and Cell Biology (Professor

Warwick

Britton

& Dr Adrew

Bean). Performing protective/challenge studies in mice to

compare

the

protective

efficacy of the DNA vaccine

with

BCG-immunised mice. Experiments need to be carried out in the ·

P3 level laboratory

which

involve aerosol tuberculosis.

8

(9)

' . .!··

Details of experimental results of the project

Objectives of the project

To construct plasmid DNA expressing mutiple T-cell epitopes of Mycobacterium tuberculosis:

towards the development of potential DNA vaccine candidates against tuberculosis.

Methodology

1. Designing of a synthetic gene {vacll) consist of multipleT-cell spitopes of M. tuberculosis using MacDNAsis and OLIGO software (Figure A).

2. Construction of vacll by assembly PCR technique (Rgure B).

3. Cloning of vacll into plasmid expression vector, pJW4303.

4. Immunization protocol for DNA vaccine candidate (Figure C).

5. lmmunogenicity studies: L TT, ELISA lFNy (Figure D) Results

1. Construction of synthetic gene( designated as vacll) consist of multipleT-cell spitopes of M.

tuberculosis and cloning of vacll into plasmid expression vector, pJW4303. The recombinant clone (designated as pJWvacfl) was secreened by double enzyme digestion (Figure E) and confinned by DNA sequencing (Figure F).

2. lmmunogenicity studies: L TT, ELISA IFNy (Table A & B) Conclusion

We have constructed one plasmid DNA expressing multipleT-cell epitopes of Mycobacterium tuberculosis (designated as pJWvacll). Preliminary studies on the immunogenicity of this plasmid has been done in mice and the results suggested that this plasmid is potentially useful as DNA vaccine candidate for tuberculosis. Further studies that to be done to evaluate this vaccine candidate indude protection efficacy in animal model.

9

(10)

,.

~ (J

•

F\gure A ONA and prote\n· sequence of vac \\ (cons\st of selected T-een epitopes of ESAT6, mtp40, 38kD and MPT64 genes of.M. tuberculosis).

•

18 2, :!6 4! $4

5' A"!G ;,.:A Gl\:; CAG ::AG TG:; }'.A:; l''l'C :;c:: GG::: ;.T:: :;A:; :<::: X~ :;cc :c:: C-c:: t.'!C

immnunmummnntmnnmmmrunnmnnrnmmuntElmlmnun:tllDtmnnu•unmums

4

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mum ^:r ^... ^•· mnnmmmmnmmmumnBII11nmmJmnummtlllUI11lllre

4

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4

•

279 BB 23"1 3C6 3:! 3<4

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•

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• •

(11)

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::1 CT CD

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(12)

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\mmun\zat\on Protocol Figure C

Plasmid DNA injected i.m. into C57BL/6J

[5 mice for pJWvacll

5 mice for the plasmid vector pJW]

n 2 weeks .

2nd injection

n 2weeks .

. 3rd injection

n 2weeks

Collect spleeri (lymphocyteS)

•

(13)

.;

Figure D

•

ELISA IFNy (using commercial kit)

.

HI!~Mtm'I!RbtJtmMM-UlfiJ!L'll-

l s

Culture S/N is added into wells coated with Ab to IFN y

JJ Washing

JJ

Enzyme Ab conjugate is the added JJ

washing

u

Enzyme substrate is added and colour reactiOn is measured by spectrophotometer (the value of IFN y is calculated

based on the standard ~urve)

'.. .

·r~-.

-

j

··'

·.

(14)

..

M ¹

-S.lkb . (vector; pJW43.03)

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882bp

(insert; Vac IT)

500bp

Figure''& Electrophoresis of recombinant clone (pJWVac II) after double enzyme digestion with Nhel and Bam HI (Lane M: 1 OObp DNA ladder, lc;tne 1:

clone pJWVacll digested with BamHI and Nhel)

1 .

. J '

(15)

Confirmation by DNA sequencing

ESAT mtp40

MTEQQWNFAGIEAAASAIQGNANPYQGVQQKWDATATELNNALQNANPASMLGT MTEQQWNFAGIEAAASAIQGNANPYQGVQQKWDATATELNNALQNANPASMLGT

38kD

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QASQRGLGEAQLGNSNANPINYEYAIVNNRQKDAATAQTLQAFLHWAITDGNAN MPT64

PPVNYQNFAVTNDGVIFFFNPGELLPEAAGPTQVLVPRSAIDSMLANANPNANP PPVNYQNFAVTNDGVIFFFNPGELLPEAAGPTQVLVPRSAIDSMLANANPNANP

Figure F.

•

(16)

I

1f1

LYMPHOCYTE TRANSFORMATION TEST (LTT)

Table A: Stmulation

Index

(SI)

of lymphocyte

cultures from

mice vaccinated

with

pJWvacii

(U)

and control mice

(K)

vaccinated

with

pJW4303 (blank plasmid ) after stimulation

with

various vacll peptides

(Sug/ml).

1

Kl K2 K3

POOLED 0.8

0.21

2.2

0./

0.6

1.0

1.0 1.0

0.7

1.0

0.3 0.5

0.7 0.4

1.1

0.11 0.6 0.9 .

0.3

1.0 2.0

38A lOug

0.04

1.02 3.3

38BSug 0.1 0.4

1.7

38B lOug

0.14

1.5 0.9

64A5ug

0.11

1.1

1.8

64A 10ug

0.13 0.5 0.9

nt 2

K4 KS

POOLED 1.8

^1.1

2.5

3.1

ESAT 1.5 0.5

5.

ESAT

^5.2 ^1.4

10.

38A5ug/ml

0.8

^0.5

12

... r:

' j • •

(17)

38B5ug

0.7

0.3

38B lOug 1.4 0.8

•

^64A5ug

0.4 0.6

•

13

'·· .. ^•.

(18)

..

ELISAIFN

Table B: Production of IFNy (ug!ml) by lymphocyte cultures from mice vaccinated

with

pJWvacll

(U)

and control mice

(K)

vaccinated with pJW4303 (blank plasmid) after stimulation

with

various vacll peptides.

Experiment 1

Kl

Pooled

peptides 500

(Sug/ml)

ESAT6

65

100

900

• • · - \.. ·• a ' • •

K2

1750

365

>1,000 150

, ^~ . , ^~·r.' i-: . • . • .

K3

1100

35

100 550

14

(19)

II

I I

. .

Appendix B: Paper presented at1 Oth MSMBB Scientific Meeting; 14-16th May 2000, Hyatt Regency Saujana, Subang (Poster PS).

"Construction of synthetic genes of Mycobacterium tuberculosis using

assembly PCR

technique''

Mustaffa Musa , Salwana Ahmad

&

Zainuf F. Zainuddin*Department of Immunology, School of Medical Sciences, School of Health

Sciences*, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan.

Abstract

The development

of

improved subunit vaccines is considered

a

high priority in the effort to control TB worldwide. Naked DNA or genetic vaccination is the current concept in vaccinology with the -use of plasmid DNA expressing certain gene. Single or multiple

gene~

of an organism

can be cloned

into

the

expression plasmids. In our ··study, assembly

PCR

technique was used to construct a synthetic genes namely, Vac Vac II. (0.82 kb). This synthetic gene were based on ESATS, mtp40, mtp64 and 38kD genes of

M. tuberculosis. Vac II

gene consist of selected T -cell epitopes of those four MTB genes. In assembly

PCR technique,

mixture

of Vac II

derived-oligos was amplified for 55 cycles {950C/30sec, 620C/30sec, 720C/30sec). The PCR product with the expected DNA band size was reamplified using vector cloning primers

(Nhe

1 and

Bam

HI primers). The final PCR products of Vac II are ready to be cloned into the expression vector.

Introduction

Tuberculosis is a major bacterial disease of humans accounting for 8 million cases of clinical disease and 3 million deaths annually. The development

of

improved subunit vaccines is considered a high priority in the effort to control TB worldwide. DNA vaccines are a new and powerful approach to the generation of the needed vaccines

(1-4). Dubbed as

the .. third revolution"

in

vaccine development, this novel method of immunization relies on plasmid expression vectors to produce the immunizing protein(s) in the vaccinated host. It involves direct Injection of plasmids, loops of DNA that contains genes for proteins produced by the organism targeted for immunity.

Once injected into the host muscle tissue, the DNA is taken up

by

host cells, which then start expressing the foreign protein. The protein serves as an antigen that stimulates immune responses.

Materials and Methods

Overlapping oligos (19 upper strand

+

19 lower strand) based on the DNA sequence of the synthetic gene (vac II - see Figure 1) were designed

using

a computer programme

(Oligo

5 & McDNAsis).

Oligos were commercially made by Operon TeChnologies Inc. and BioBasic Inc. through Bionsyntech Sdn. Bhd. Assembly PCR was

10

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DNA and protein sequence of vac U

9 18 27 36 45 54 ~:11 ~96 40S 414 42~ '32

5' ~~~~~~~~~~~~~~~~~~ =AC C~G AA: 3CA AA~ CCA ~~~ CT3 GGC GA3 AAC GGC AAC G3C G3C ArG G~G ACA

---

^~--~---

--- --- --- --- --- --- --- --- --- --- --- --- ---

Ul '59 'Ee 477

3G: ~GC GT3 G~C ~AC ATC G3C ArC ~CC rr:

63 72 81 90 99 lOB

~ GGC A'/>.T G:A Mi' CCA TAC CAG GGC GTG CAG CAG ».G 'IGG GAC GCC N:A GCC

'93 3:H 3D S22 S3: !'1'0

=AG 3C: rc: CAG CG: GGC CTG GGC GA3 G~C CA3 :'l'G G3C 1\AC TCC AAT 3CG AA'I

ll u

117 126 135 144 153 162

M:.A ~ erG Ar..r; 'J1.N:. OC.C erG CAG /AX Gt"..A A'/>.T CCA GCC ':OCC ATG CTG GGC AO\

Sf9 33e 567 S76 583 ~94

=cG A~~ AA: ~A= GAG ~AC G:= Arc GT3 AAC AAC :::;c .CA3 1\AG 3AC ::;c: 3CC A:A

--- --- --- --- --- --- --- --- --- --- --- --- ---

~MW!llmffPIJMtWH!OO-a(lft1lfm!I!Qilt!11!!S!!JmDPNM·MBi

l 11 180 189 198 207 216 6:l3 6:2 e2: E3:: 639 64E

GTG .ru:A. AF>~ TCC CCC GGC GTG CCC GGC GGC GCC GI'G GGC GOC G'l'G M:A TGG TO: GCC CAG ACA C~G CAG GCC ~'I: CT3 CAC TG3 GCC ATC ACA GAC G3C AAT GCA AA'I

637 6E6 61~ ce4 c93 7C2

=cA CC~ 3:G AA= rA: CA3 AA: ~TC G=c G'I3 ACA AAC GAC G3C G~G AT: T~C r~~

--- --- --- ---

^~--

--- --- --- --- --- --- --- --- --- ---·---

225 234 243 252 261 270 5ftiiRiliilll!liiHIIIJDIIP'JIIIfUIISIUIIIiBimmmmmlfilmllmmt:illllHDnnmnmunmDHmmmnnmmmmlBllHilll

'l:l 1.20 729 73:1 , , , . 7~c

T~C AA: CC: 3G= 3AG c:3 C~G c:c GA3 G:C GCC GGC CCC ACA CAG 3TG =~G 3~G

---

iilUIJIJO,nmmiiUIOOHIIMimiH!IIIFJIIIIHIIIDDJIHIDmiHiffiHIIIIfiP.iUHDHiilliJIIIIUruHIIllfill11lll11~lutllllh1111DiimmJIIIUIUIJi!_ ^---

--- --- --- ---

2'79 289 297 306 315 324 •

CTG CTG G::C G1'G C'l'G PC..'A,.. GCC GCC CCC CTG erG C1'G ~ MT OCA AM CCA ~

763 ' ' 4 '33 792 3o: e1c

t~AN

:?0

^=ccc::;: rc: 3C= A1: GAC ~~~ AT3 CT3 GCC AAC G=A AA~ =cA AA~ 3CG AA~ CCG

h If u A N

333 342 351 360 369 378

~~~~~~~CAG~~~~~~~~~~ 819

---

^---

---

^-~-

--- --- --- --- --- --- --- --- --- --- ---

^---

---

^--- AAT GCA AAT CCA 3'

f

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•

performed

with

slight modification according to the method described

by

Stemmer eta/. (5). This technique involves

2

steps (Figure

2);

1st step: ovalapping

oligos were mixed in PCR

reaction buffer, amplified for

55

cycles (950C/30sec, 620C/30sec, 720C/30sec). 2nd Step:

PCR product of the first step (with the expected band size) was re- PCR (950C/30sec, 620CJ30sec, 720C/30sec) using vector cloning primers (Nhe11BamH1 primer) for 55 cycles. The PCR products were analysed by agarose gel electrophoresis.

Results and Discussion .

AssembJy PCR (1st step PCR)

using the

Vac II

oligos showed smear band when ~nalysed with agarose gel electrophoresis (Figure 3A).

From the first step product as a template, we did the 2nd step PCR using the cloning primers (Nhe I dan Bam H 1). We managed to get the correct size of the product which was 0.82kb (Vac II) (Figure 38).

The synthetic gene is being cloned into plasmid expression yector (pJW4303) for further analysis. Such analysis included DNA sequencing to determine the gene sequences and whether the gene is in frame or not. The results showed that assembly PCR technique employed in this study were able to generate selected genes of M.

tuberculosis. It shows that assembly PCR technique is a useful toof to generate synthetic gene or multiple genes for the development of

DNA

vaccine.

References

1. Robinson HL, Hune LA, Webster RG (1993). Protection against a lethal influenza

virus

challenge by immunization with a

haemaglutinin-expressing

plasmid DNA. Vaccine

11:957-960.

2. Ulmer JB,

Donnelly

JJ, Parker SE,

Rhodes

GH et al

(1993).

Heterologous protection against influenza by

injection of

DNA encoding a viral protein.

Science

259:1745-1749.

3.

Wang 8, Ugen

Kt,

Srikantan V, et

al

(1993) . Gene inoculation generates

immune responses

against human

immunodeficiency

virus type l.lmmunology 90: 4146-4160.

4.

Fynan EF,

Webster

RG,

Fufler

DH, haynes JR,Santoro

JC,Robinson HL (1993) DNA

vaccines:

Protective

immunizations by parental, mucosal, and gene-gun inoculations. Immunology 90:11478-11482.

5. Stemmer eta/. (1995) Single-step assembly of a gene and entire plasmid from large numbers of oligodeoxyribonucleotides.

Gene.164:(49-53).

11

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•

Overlapping ~

o llgos

Nho I Cloning prim ar

2 n s1 t1t g P Q A w ltb glppln g prim trp:

DI5°C/30ooa

ea•c 130 •• c 72°CI30soo (3D oycl••l

DNA sequence ofVac I orvac II

m he

1 Jt etpp p C B • 115 ¹C/30 ••c

112¹C/3Da•o 72'CI:tDactc (55 C)'CIOI)

Electrophorosls to vlauallzo

C u t a n d c lo n a In to oxpreaalon voctor

B a 11 H I c lo n In g prim or

B·

F lg u re 2,: A Bee m b ly PC R to Den era te V a o I & V a c II gene a

.•

..

....

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\

f

A. 1st step PCR

M

600__..

B. 2"d step PCR (cloning primers)

2702 _ _ ___,.,...

1500 _ _ ___,.,...

600 ...

100 ...

M 1

~

IOObp Vac ll marker

1

Lane M - 1 OObp marker

Lane 1 - PCR product for Vac ll

2072__..

1500 ____..

600_ _ . .

(2) 2"d round

M 1

...~1--- 0.82kb

lOObp

marker Vac IT

Figure 3: Analysis of the PCR product by agarose gel electrophoresis. A. 1st step PCR

i (l),(2). 2"'

step

PCR

\