Otwarty dostęp

Cloning and expression of NS3 gene of Pakistani isolate type 2 dengue virus

Farkhanda Yasmin
Farkhanda Yasmin
, 
Tahir Yaqub
Tahir Yaqub
, 
Muhammad Idrees
Muhammad Idrees
, 
Wasim Shahzad
Wasim Shahzad
, 
Abu Saeed Hashmi
Abu Saeed Hashmi
, 
Kiran Aqil
Kiran Aqil
, 
Nadia Mukhtar
Nadia Mukhtar
, 
Muhammad Yasir Zahoor
Muhammad Yasir Zahoor
, 
Naeem Akhtar
Naeem Akhtar
 oraz 
Sajid Umar
Sajid Umar
   | 30 mar 2018
Journal of Veterinary Research's Cover Image
O artykule
Poprzedni artykuł
Następny artykuł
Zacytuj
Data publikacji: 30 mar 2018
Zakres stron: 17 - 26
Otrzymano: 19 wrz 2017
Przyjęty: 08 mar 2018
DOI: https://doi.org/10.2478/jvetres-2018-0003
Słowa kluczowe
© 2018 F. Yasmin et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Fig. 1

PCR amplification of dengue NS3 gene. Lane M – 1Kb marker; lanes 1-3 – amplified NS3 product
PCR amplification of dengue NS3 gene. Lane M – 1Kb marker; lanes 1-3 – amplified NS3 product

Fig. 2

The cloning of fragment containing NS3 region in pCR 2.1 TOPO
The cloning of fragment containing NS3 region in pCR 2.1 TOPO

Fig. 3

(a) Restriction and digestion of NS3 encoding TA vectors. b) Cloning PCR
(a) Restriction and digestion of NS3 encoding TA vectors. b) Cloning PCR

Fig. 4

Map of pcDNA3.1 mammalian expression vector
Map of pcDNA3.1 mammalian expression vector

Fig. 5

PCR amplification of NS 3 gene with restriction site specific primers. Lane 1 M – 1 kb marker, lanes 2–4 – NS3 (1,904 bp)
PCR amplification of NS 3 gene with restriction site specific primers. Lane 1 M – 1 kb marker, lanes 2–4 – NS3 (1,904 bp)

Fig. 6

Double-digested pcDNA3.1 mammalian expression vector. Lane M – 1kb marker; lanes 1 and 2 – digested pcDNA 3.1
Double-digested pcDNA3.1 mammalian expression vector. Lane M – 1kb marker; lanes 1 and 2 – digested pcDNA 3.1

Fig. 7

Double-digested amplified NS3 gene of dengue virus. Lane M – 1kb marker; lanes 1–3 – double digested NS3 gene
Double-digested amplified NS3 gene of dengue virus. Lane M – 1kb marker; lanes 1–3 – double digested NS3 gene

Fig. 8

Colony PCR (gene-specific) screening of NS3 encoding region in mammalian expression vector. Lanes 1–2 – positive NS3 encoding clones; lane M – 1kb ladder
Colony PCR (gene-specific) screening of NS3 encoding region in mammalian expression vector. Lanes 1–2 – positive NS3 encoding clones; lane M – 1kb ladder

Fig. 9

Restriction and digestion of NS-3 encoding mammalian expression vector. Lane M – 1kb marker; lanes 1–3 – positively digested plasmids
Restriction and digestion of NS-3 encoding mammalian expression vector. Lane M – 1kb marker; lanes 1–3 – positively digested plasmids

Fig. 10

pcDNA3.1 BglII linearised plasmid and empty vector. Lane M – 1kbM ladder; lanes 1 and 2 – linearised pcDNA3.1; lane M – 1kb ladder; lane 1 – linearised pcDNA3.1/NS3
pcDNA3.1 BglII linearised plasmid and empty vector. Lane M – 1kbM ladder; lanes 1 and 2 – linearised pcDNA3.1; lane M – 1kb ladder; lane 1 – linearised pcDNA3.1/NS3

Fig. 11

NS3 expressing cell line characterised by RT-PCR
NS3 expressing cell line characterised by RT-PCR

Primers used for amplification and expression of dengue virus NS3 gene. Restriction sites were added at the start of primers

No. Genes Primer sequence
1 NS3F-1 GCAAGCTTGCCATGGCCGCTGGAGTATTGTCGGC
2 NS3F-2 GCGGATCCGCCATGGCCGCTGTATTGTCGGC
3 NS3R GCGCGGCCGCTTTCTTCCACTGCAAACTCTTTGTTC

List of bacterial strains used in the present study

No. Bacterial strain Genotype and description Source
1 E. coli DH 5 α F’, φ80d/lacZ.M15, recA1, endA1, gyrA96, thi-1, hsdR17(rK-, mK+), supE44, relA1, deoR, .(lacZYAargF) U169; CEMB culture Collection
2 E. coli DH 5 α top10 F- mcrA .(mrr-hsdRMS-mcrBC) ö80lacZ.M15 .lacX74 recA1 araD139 CEMB culture Collection

List of primers used for amplification of dengue virus NS3 gene

No. Primer Name Primer Sequences Primer position within N2L1 sequence
1 NS3-1-OF CAGGACTTTTCCCCGTATCA 4419-4438
2 NS3-1-IF AACAACGGGCTGGAGTATTG 4482-4501
3 NS3-1-SEQ-F CCAGGTCTTGGCATTAGAGC 4774-4793
4 NS3-2-OF TCACAGACCCAGCAAGCATA 5352-5371
5 NS3-2-IF AGCAGAGACCCATTTCCTCA 5450-5469
6 NS3-2-SEQ-IF GGCAGAAATGGGTGCTAACT 5719-5738
7 NS3-1-OR GGAACTCTGGACATGAGTGGGT 5541-5520
8 NS3-1-IR TTGAGGAAATGGGTCTCTGC 5451-5470
9 NS3-2-OR AGCATGATTGTACGCCCTTC 6456-6475
10 NS3-2-IR TGGAAGCCTACCCATTTCTG 6366-6385

pcDNA3.1 vector specific primer sequences

Name Primer sequence
T7 TAATACGACTCACTATAGGG
BGH TAGAAGGCACAGTCGAGG
eISSN:
2450-8608
Język:
Angielski
Częstotliwość wydawania:
4 razy w roku
Dziedziny czasopisma:
Life Sciences, Molecular Biology, Microbiology and Virology, other, Medicine, Veterinary Medicine
Kanał RSS czasopisma