携带小鼠白介素-4截短型突变体基因的5型腺相关病毒载体的构建及外源蛋白的体外表达
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摘要
目的:制备携带碳端结构域缺失的小鼠白介素-4(Interleukin-4,IL-4)基因突变体的5型重组腺相关病毒(recombinant adeno-associated virus, r AAV)并在细胞水平检测其介导的外源蛋白表达情况。方法:通过分子克隆技术构建携带小鼠IL-4碳端22个氨基酸缺失的突变体的表达质粒pSNAV-mIL-4ΔC22,三质粒共转染法制备重组的5型AAV病毒,体外感染人支气管上皮样细胞系16HBE和BEAS-2B,并通过Western blot和ELISA检测外源蛋白表达。结果:DNA测序表明构建的小鼠IL-4碳端第118位氨基酸位点处截短的突变体基因表达序列正确无误,制备的重组病毒载体r AAV5-mIL-4ΔC22滴度约为3×10~(11)vg/m L。r AAV5-GFP感染16HBE和BEAS-2B细胞后36小时开始可见持续稳定的荧光蛋白表达,重组病毒r AAV5-mIL-4ΔC22感染16HBE和BEAS-2B细胞后外源蛋白在培养上清中呈分泌型表达。结论:本研究成功构建了携带小鼠IL-4碳端结构域缺失型突变体的AAV表达质粒并制备了重组病毒r AAV5-mIL-4ΔC22,该病毒可有效转染16HBE和BEAS-2B细胞并介导外源基因分泌表达截短型小鼠IL-4突变体蛋白。
Objective: To prepare recombinant adeno-associated virus harboring truncated murine interleukin-4 mutant gene and detect the exogenous protein expression in vitro. Methods: An m IL-4 antagonistic mutant DNA expression plasmid pSNAV-mIL-4ΔC22 was constructed through gene clone technology, a recombinant virus vector r AAV5-mIL-4ΔC22 was prepared by three plasmids co-transfection, human bronchoid epithelioid cell line 16 HBE and BEAS-2 B were transfected with recombinant AAV5 vectors, then the expression of foreign protein was detected by Western blot and ELISA. Results: The mutant gene m IL-4ΔC22 was verified by DNA sequencing and the titer of virus vector r AAV5-mIL-4ΔC22 is about 3×10~(11)vg/m L. 16 HBE and BEAS-2 B expressed GFP lastly and stably when 36 hours after receiving r AAV-GFP trensfection. Conclusions: Our study constructed recombinant vector r AAV5-mIL-4ΔC22 encoding murine IL-4 antagonistic mutant protein deleting the C-terminus region, the virus vector effectively transfected 16 HBE and BEAS-2 B, and mediated exogenous protein secretory expression in vitro.
Objective: To prepare recombinant adeno-associated virus harboring truncated murine interleukin-4 mutant gene and detect the exogenous protein expression in vitro. Methods: An m IL-4 antagonistic mutant DNA expression plasmid pSNAV-mIL-4ΔC22 was constructed through gene clone technology, a recombinant virus vector r AAV5-mIL-4ΔC22 was prepared by three plasmids co-transfection, human bronchoid epithelioid cell line 16 HBE and BEAS-2 B were transfected with recombinant AAV5 vectors, then the expression of foreign protein was detected by Western blot and ELISA. Results: The mutant gene m IL-4ΔC22 was verified by DNA sequencing and the titer of virus vector r AAV5-mIL-4ΔC22 is about 3×10~(11)vg/m L. 16 HBE and BEAS-2 B expressed GFP lastly and stably when 36 hours after receiving r AAV-GFP trensfection. Conclusions: Our study constructed recombinant vector r AAV5-mIL-4ΔC22 encoding murine IL-4 antagonistic mutant protein deleting the C-terminus region, the virus vector effectively transfected 16 HBE and BEAS-2 B, and mediated exogenous protein secretory expression in vitro.
引文
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[28]Hurdayal R,Brombacher F.Interleukin-4 Receptor Alpha:From Innate to Adaptive Immunity in Murine Models of Cutaneous Leishmaniasis Front[J].Immunol,2017,(8):1354
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[2]Holgate ST,Wenzel S,Postma DS,et al.Asthma[J].Nat Rev Dis Primers,2015,1:15025
[3]Bollmeier S:Clinical updates on the management of asthma[J].Am JManag Care,2017,23(1 Suppl):S3-S11
[4]Walsh GM.Anti-IL-4/-13 based therapy in asthma[J].Expert Opin Emerg Drugs,2015,20(3):349-352
[5]Tabatabaian F,Ledford DK.Omalizumab for severe asthma:toward personalized treatment based on biomarker profile and clinical history[J].J Asthma Allergy,2018,11:53-61
[6]Shamji MH,Durham SR.Mechanisms of allergen immunotherapy for inhaled allergens and predictive biomarkers[J].J Allergy Clin Immunol,2017,140(6):1485-1498
[7]Darveaux J,Busse WW.Biologics in asthma--the next step toward personalized treatment[J].J Allergy Clin Immunol Prac 2015,3(2):152-160;quiz 161
[8]Walsh GM.Biologics targeting IL-5,IL-4 or IL-13 for the treatment of asthma-an update[J].Expert Rev Clin Immuno,2017,13(2):143-149
[9]Doran E,Cai F,Holweg CTJ,et al.Interleukin-13 in Asthma and Other Eosinophilic Disorders[J].Front Med(Lausanne)2017,4:139
[10]Wang T,Secombes CJ.The evolution of IL-4 and IL-13 and their receptor subunits[J].Cytokine,2015,75(1):8-13
[11]Ranasinghe C,Trivedi S,Wijesundara DK,et al.IL-4 and IL-13 receptors:Roles in immunity and powerful vaccine adjuvants[J].Cytokine Growth Factor Rev,2014,25(4):437-442
[12]Gandhi NA,Pirozzi G,Graham NMH.Commonality of the IL-4/IL-13 pathway in atopic diseases[J].Expert Rev Clin Immunol,2017,13(5):425-437
[13]Karo-Atar D,Bitton A,Benhar I,et al.Therapeutic Targeting of the Interleukin-4/Interleukin-13 Signaling Pathway[J].In Allergy and Beyond.BioDrugs,2018
[14]La Porte SL,Juo ZS,Vaclavikova J,et al.Molecular and structural basis of cytokine receptor pleiotropy in the inter leukin-4/13 system[J].Cell,2008,132(2):259-272
[15]Walter MR,Cook WJ,Zhao BG,et al.Crystal structure of recombinant human interleukin-4[J].J Biol Chem,1992,267(28):20371-20376
[16]Zabner J,Seiler M,Walters R,et al.Adeno-associated virus type 5(AAV5)but not AAV2 binds to the apical surfaces of airway epithelia and facilitates gene transfer[J].J Virol,2000,74(8):3852-3858
[17]Sumner-Jones SG,Davies LA,Varathalingam A,et al.Long-term persistence of gene expression from adeno-associated virus serotype 5in the mouse airways[J].Gene Ther,2006,13(24):1703-1713
[18]Guggino WB,Benson J,Seagrave J,et al.A Preclinical Study in Rhesus Macaques for Cystic Fibrosis to Assess Gene Transfer and Transduction by AAV1 and AAV5 with a Dual-Luciferase Reporter System[J].Hum Gene Ther Clin Dev,2017,28(3):145-156
[19]Boutin S,Monteilhet V,Veron P,et al.Prevalence of serum Ig G and neutralizing factors against adeno-associated virus(AAV)types 1,2,5,6,8,and 9 in the healthy population:implications for gene therapy using AAV vectors[J].Hum Gene Ther,2010,21(6):704-712
[20]Halbert CL,Miller AD,Mc Namara S,et al.Prevalence of neutralizing antibodies against adeno-associated virus(AAV)types 2,5,and 6 in cystic fibrosis and normal populations:Implications for gene therapy using AAV vectors[J].Hum Gene Ther,2006,17(4):440-447
[21]Martini SV,Fagundes SS,Schmidt AC,et al.Does the use of recombinant AAV5 in pulmonary gene therapy lead to lung damage?[J].Respir Physiol Neurobiol,2009,168(3):203-209
[22]Bevaart L,Aalbers CJ,Vierboom MP,et al.Safety,Biodistribution,and Efficacy of an AAV-5 Vector Encoding Human Interferon-Beta(ART-I02)Delivered via Intra-Articular Injection in Rhesus Monkeys with Collagen-Induced Arthritis[J].Hum Gene Ther Clin Dev,2015,26(2):103-112
[23]Srivastava A,Carter BJ.AAV Infection:Protection from Cancer[J].Hum Gene Ther,2017,28(4):323-327
[24]Loo SL,Wark PAB.Recent advances in understanding and managing asthma[version 1;referees:2 approved]F1000Research[M].2016,5(F1000 Faculty Rev):2052
[25]Walter MR,Cook WJ,Zhao BG,et al.Crystal Structure of Recombinant Human Interleukin-4[J].J Biol Chem,1992,267(28):20371-20376
[26]Kelly-Welch AE,Hanson EM,Boothby MR,et al.Interleukin-4 and interleukin-13 signaling connections maps[J].Science,2003,300(5625):1527-1528
[27]David Richter,Ignacio Moraga,Hauke Winkelmann,et al.Ligand-induced type II interleukin-4 receptor dimers are sustained by rapid re-association within plasma membrane microcompartments[J].Nat Commun,2017,14(8):15976
[28]Hurdayal R,Brombacher F.Interleukin-4 Receptor Alpha:From Innate to Adaptive Immunity in Murine Models of Cutaneous Leishmaniasis Front[J].Immunol,2017,(8):1354
[29]Junttila IS.Tuning the Cytokine Responses:An Update on Interleukin(IL)-4 and IL-13 Receptor Complexes[J].Front Immunol,2018,7(9):888
[30]Binita Kane,Stephen J Fowler,Rob Niven.Refractory asthma-beyond step 5,the role of new and emerging adjuvant therapies[J].Chron Respir Dis,2015,12(1):69-77
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