2011 9 1 77 SLC26A4 HEK 293 1 2 1 1 1 1 (100853) 2 ( 050051) SLC26A4 (pegfp enhanced green fluorescent protein) SLC26A4 pegfp-slc26a4 HEK293 pegfp-slc26a4 HEK293 - (RT-PCR) SLC26A4 mrna Western Blot ( ) Pendrin SLC26A4 GenBank SLC26A4 pegfp-slc26a4 HEK293 24 RT-PCR SLC26A4 Western Blot 87 kda SLC26A4 pegfp HEK293 SLC26A4; ; R349.6 R394.8 A 1672-2922(2011)01-077-05 The construction of wild-type SLC26A4 gene expression vector and the expression in HEK293 cells WU Jian 1 ZHANG Guo-zheng 2 YANG Shi-ming 1 DAI Pu 1 YUAN Yong-Yi 1 1 Department of Otolaryngology Head and Neck Surgery Institute of Otolaryngology Chinese PLA Genernal Hospital Beijing 100853 China 2 Department of Otolaryngology Third Hospital of Hebei Medical University Shijiazhuang 050051 China Corresponding author: YUAN Yong-yi Email: linc301@yahoo.com Abstract Objective To construct a eukaryotic expression vector containing the wild-type SLC26A4 gene and green fluorescent protein gene for clinical deafness diagnosis and prenatal diagnosis about SLC26A4 -related deafness. Methods Using the Gene recombination and restriction enzyme digestion and gene sequencing to construct and identify the eukaryotic expression plasmid of pegfp-slc26a4. Using fluorescence microscope to observe the expression of transfected HEK293 cells by liposome using reverse transcription-polymerase chain reaction (RT-PCR) to detect the mrna and using Western Blot to detect SLC26A4 protein expression. Results The positive recombinant gene contain the SLC26A4 fragment and the sequence were the same sequence as on the GenBank. After being transfected Into HEK293 cells The green fluorescent can be observed and The SLC26A4 bands can be amplified. The 87kDa-sized protein was detected by Western Blot. Conclusion we successfully constructed the eukaryotic expression vector containing wild-type SLC26A4 and EGFP gene and the vector can express in mammalian HEK293 cell lines. Key Words SLC26A4; Plasmid; Expression : (2009B34) : 2008 (30801285); 1000 50% : : Email: linc301@yahoo.com
78 Chinese Journal of Otology Vol. 9 No.1 2011 3 μl PCR PCR [1] Yuan [2] 2 360 bp 54.93% 1.2 PCR SLC26A4 13.73% GJB2 : 10 5 μl 100 BSA Pendred 1.3 PCR SLC26A4 780 100 ng PCR 100 ng T4 DNA 2 Pendrin Pendrin SO - 4 Ligase (T4 DNA ) 4 HCO 3- I - Cl - 1.4 Pendrin 1.4.1 DH5α SLC26A4 200 μl HEK293 (human enbryonic kidney cell line ) 30 min 42 87 kda 1 1.1 SLC26A4 1.1.1 150 μl RNA 20 mmol / L MgSO 4 AMP ( ) cdna PCR : LB Xho -F:5' -CCGCTCGAGATGGCAGCGCCAG GCGGCAG-3' Kpn -R:5' -GGGGTACCGTGGATGCAAGTGT 37 16 h DNA pegfp (enhanced green fluorescent protein)-slc26a4 ACGCATAG-3' 1.4.2 NCBI GenBank SLC26A4 cdna Primer Premier Kpn( 1 / 10) 20 μl 5.0 37 2 h 1.1.2 PCR ( ) 1 μg Invitrogen Invtrogen PCR : 10 Pfx50 PCR Mix ( MgCl 2 ) 5 μl 10 1.5 pegfp-slc26a4 HEK293 mmol / L dntp ( ) Mix 1.5 μl Primer Mix 1.5 μl DNA 3 μl Pfx50 DNA 1.5.1 3 μl 50 μl 6 : 94 30 s 94 30 s 55 30 s 72 3 s 72 10 s 4 30 1 Xho Kpn (bovin serum alburmin ) 0.5 μl SLC26A4 pegfp-n1 PCR 2 μl Xho 1 μl Pendred Kpn 1 μl 20 μl 37 1 h 4% ~ 10% Pendred DNA ( pegfp-n1 ) 2 μl 90 s l ~ 2 min 800 μl LB 37 37 45 min 0.5 ~ 1 μg Xho 0.5 10 5 / ml HEK293 80% DNA Lipofectamine TM 2000
2011 9 1 79 (Invitrogen) 1 μg 3μl 2 SDS ( ) DMEM (Dulbecco's Modified Eagle Medium) 100 3 min ( ) 100 μl 20 min SDS-PAGE (SDS- ) DNA- Lipofectamine TM 2000 PVDF ( ) 797.5 μl 6 h Pendrin 1 2000 37 2 h 1 000 μl 24 h 4% TBS-Tween (Tween 20 Tris ) 40 min 10 min 3 ( ) IgG 1 2000 50 min TBS-Tween 1.5.2 RT-PCR ( - ) Western 10 min 3 Blot ( ) pegfp-slc26a4 24 h 0.25% 800 r / min IgG (1 2000 ) 1.5 ml Eppendorf RT-PCR Western Blot 1.5.2.1 RT-PCR SLC26A4 / β-actin Trizol (Invitrogen) RNA 3 cdna 2 RT-PCR (Invitrogen) PCR (500 ~ 750 bp) mrna PCR : 95 5 min 95 45 s 58 45 s 72 60 s 72 5 min 40 : 5'-GAATTCCACCATGGCAGCCG- GGAGCATCA-3' : 5'-TCTAGATCAGCTCTTAGCAGA- CATTGG-3' 1.5.2.2 Western Blot X 1 2000 β-actin (β- ) Labworks4.5 2.1 Xho Kpn 2360 bp 1 2.2 pegfp -SLC26A4 NCBI GenBank NM_000441.1 2.3 pegfp- SLC26A4 HEK293 HEK293 2A 2B Western Blot 1 1 DL2000 Marker 2 pegfp-slc26a4 2 pegfp-slc26a4 HEK293 RT-PCR Western Blot a. HEK293 ( DAPI ); b. pegfp-slc26a4 ; c. RT-PCR : 1 DL5000 Marker 2 pegfp-slc26a4 cdna PCR 3 266 bp β-actin; d. Western Blot : 1 87 kda 2 42~43kDa β-actin 2 a b 50 μm
80 Chinese Journal of Otology Vol. 9 No.1 2011 RT-PCR mrna (15 / 98) Belguith-Maalej [10] 52 2C 2D ( 22 (Graves' disease) 87 kda 3 Pendred Pendred 1896 Pendrin Everett [3] 1996 (autoimmune thyroid diseases) SLC26A4 1996 Griffith [4] SLC26A4 Pendrin (LVAS) 1999 Abe [5] LVAS 7q31 Usami [6] SLC26A4 (Pendred ) SLC26A4 LVAS SLC26A4 7q31 21 780 [7] 0 ~ 3 130 130 54 12 7 85 65.4% (85 / 130); 23 ; 13 2 PCR SLC26A4 : 42 SLC26A4 21 1 32.3%(42 / 130); 6 GJB2 Pendrin 4.6% (6 / 130); 36.9% pegfp-slc26a4 HEK293 (SLC26A4 GJB2 ) HEK293 [8] 1552 197 kda pegfp-slc26a4 S7-2A>G 83 S7-2 A>G 114 S7-2A>G S7-2A>G 12.69%(197 / 1552) 114 S7-2A>G PCR : (1) 78 36 ; (2) PCR ; 1552 S7-2A>G (3) PCR S7-2A>G PCR 161 10.37% (161 / 1552) 78 SLC26A4 S7- pegfp-n1 2A>G 190 19 10 17 15 11+12 14 3 ; 21 S7-2 A>G EGFP Kozak [12] 2% [9] 98 pegfp-slc26a4 EGFP N 48 31 19 GJB2 29.6% (29 / 98) mtdna A1555G 4.08%(4 / 98) SLC26A4 15.31% 11 5 3 8 1 2 ) SLC26a4 Kim [11] Slc26a4 SLC26A4 PDS Pendrin Pendrin N C / 2A 2D Western Blot 87 RT-PCR SLC26A4 mrna 2 360 bp SLC26A4 EGFP
2011 9 1 81 HEK293 87 kda associated with enlarged vestibular aqueduct is caused by PDS mutations. Hum Genet 1999 104(2): 188-192. SLC26A4 7. 130. 2009 44 (3): 177-181. 1. SLC26A4 S7-2 A>G.. 2005 3(1): 62-64. 2 Yuan Y You Y Huang D et al. Comprehensive molecular etiology analysis of nonsyndromic hearing impairment from typical areas in China. J Transl Med 2009 7: 79. 3 Everett LA Glaser B Beck JC et al. Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS). Nat Genet 1997 17(4): 411-422. 4 Griffith AJ Arts A Downs C et al. Familial large vestibular aqueduct syndrome. Laryngoscope 1996 106(8): 960-965. 5 Abe S Usami S Hoover DM et al. Fluctuating sensorineural hearing loss associated with enlarged vestibular aqueduct maps to 7q31 the region containing the Pendred gene. Am J Med Genet 1999 82(4): 322-328. 6 Usami S Abe S Weston MD et al. Non -syndromic hearing loss 8. 1552 2009 44(6): 449-454. 9.. 2010 27(5): 530-534. 10 Belguith -Maalej S Rebuffat SA Charfeddine I et al. SLC26A4 expression among autoimmune thyroid tissues. Immunobiology 2010 216(5): 571-578. 11 Kim HM Wangemann P. Failure of fluid absorption in the endolymphatic sac initiates cochlear enlargement that leads to deafness in mice lacking pendrin expression. PLoS One 2010 5 (11): e14041. 12 CLONTECH Laboratories Inc. pegfp -N1 Vector Information [EB / OL]. (1999-03 -19) [2011-01 -05]. http://www.synthe sisgene.com/vector/pegfp-n1.pdf ( :2010-12-20) Richard Salvi 2008 25