HCMV主要立即早期蛋白IE_286相互作用蛋白的筛选
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摘要
目的:本研究拟用Pull-Down法从12周龄人胎脑组织中“钓取”与IE_286相互作用的宿主蛋白,以探讨IE_286在HCMV致CNS畸形中的作用机制。
方法:(1)以pBT-IE_286质粒为模板,PCR法扩增IE_286编码基因UL122,BamHI/NotI双酶切,克隆入载体pGEX-4T-1相应克隆位点,连接,转化入大肠杆菌BL21中,并经PCR、限制性酶切和测序鉴定。(2)重组质粒转化菌于30℃培养条件下经IPTG诱导表达GST-IE_286融合蛋白,融合蛋白经谷胱甘肽-Sepharose 4B柱层析提取纯化,同时对含GST基因的pGEX-4T-1空载体进行诱导表达及纯化,作为对照。(3)取12周龄胎儿脑组织,将其研磨成匀浆,用Pull-Down法筛选与GST-IE_286蛋白相互作用的蛋白。(4)获得稳定表达的蛋白条带,采用四极杆飞行时间串联质谱仪进行蛋白分析。
结果:(1)成功构建pGEX-4T-1/IE_286诱饵质粒,测序结果与GenBank中序列比对无突变。(2)成功表达并纯化GST-IE_286蛋白及GST蛋白,GST-IE_286蛋白约为92kDa,GST蛋白为26kDa,与预期蛋白大小一致。(3)利用Pull-Down法,从胎儿脑组织中“钓取”了4个IE_286相互作用的蛋白:Ⅱ型角蛋白(K2C1)、Ⅰ型角蛋白(K1C10)、组蛋白H2B以及波形蛋白。
结论:HCMV IE_286可与12周龄人胎脑组织细胞的Ⅱ型角蛋白(K2C1)、Ⅰ型角蛋白(K1C10)、组蛋白H2B以及波形蛋白相互作用,但其相互作用的生物学意义及其在HCMV感染致胚胎CNS畸形发育中的作用有待进一步研究。
Objective: Proteins interacting with IE_286 from the brain tissue of 12-week-old embryo were screened by Pull-Down assay, to investigate the role of IE_286 in CNS anomalies induced by HCMV congenital infection and its possible mechanisms.
Methods: (1) UL122 CDS encoding IE_286 was amplified by PCR with pBT-IE_286 plasmid as template, and then the amplification product was digested with restriction endonucleases BamHI and NotI, and cloned into the corresponding sites on bait plasmid pGEX-4T-1. The recombinant was transformed into E. coli BL21. Followed by PCR, restriction and DNA sequence analysis. (2) GST-IE_286 fusion protein was expressed in 30℃for 3-4 hours under 1mM IPTG induction and purified by affinity column chromatography; GST protein was expressed and purified as negative control. (3) Fetal brain tissue from 12-week-old embryo was taking out and grinding into homogenate. The proteins interacting with IE_286 were screened from lysates by Pull-Down assay. (4) Cut the stably expressive protein bands and analysed the proteins with Quatropde-Time of Flight Mass spectrometer.
Results: (1) The pGEX-4T-1/IE_286 bait plasmid was constructed successfully. The result of DNA sequence analysis was no mutation compared with the sequence in GenBank. (2) GST- IE_286 fusion protein and GST protein were expressed and purified successfully. The protein molecular weight of GST- IE_286 fusion protein was 92kDa, and GST protein was 26kDa, consistent with the expected protein molecular weight. (3) The fetal brain protein which involved with IE_286 has been found by using the method of Pull-Down assay, after mass spectrometry detection, there are four kinds of protein: Keratin, type II cytoskeletal 1, Keratin, type I cytoskeletal 10, Histone, H2B type 1-B and Vimentin.
Conclusions: HCMV IE_286 may have interaction with four kinds of protein from 12-week-old embryo: Keratin, type II cytoskeletal 1, Keratin, type I cytoskeletal 10, Histone, H2B type 1-B and Vimentin, however, the biological significance and the role of these interaction in fetal congenital anomalies of the central nervous system (CNS) which caused by infection of HCMV need to be further studied.
方法:(1)以pBT-IE_286质粒为模板,PCR法扩增IE_286编码基因UL122,BamHI/NotI双酶切,克隆入载体pGEX-4T-1相应克隆位点,连接,转化入大肠杆菌BL21中,并经PCR、限制性酶切和测序鉴定。(2)重组质粒转化菌于30℃培养条件下经IPTG诱导表达GST-IE_286融合蛋白,融合蛋白经谷胱甘肽-Sepharose 4B柱层析提取纯化,同时对含GST基因的pGEX-4T-1空载体进行诱导表达及纯化,作为对照。(3)取12周龄胎儿脑组织,将其研磨成匀浆,用Pull-Down法筛选与GST-IE_286蛋白相互作用的蛋白。(4)获得稳定表达的蛋白条带,采用四极杆飞行时间串联质谱仪进行蛋白分析。
结果:(1)成功构建pGEX-4T-1/IE_286诱饵质粒,测序结果与GenBank中序列比对无突变。(2)成功表达并纯化GST-IE_286蛋白及GST蛋白,GST-IE_286蛋白约为92kDa,GST蛋白为26kDa,与预期蛋白大小一致。(3)利用Pull-Down法,从胎儿脑组织中“钓取”了4个IE_286相互作用的蛋白:Ⅱ型角蛋白(K2C1)、Ⅰ型角蛋白(K1C10)、组蛋白H2B以及波形蛋白。
结论:HCMV IE_286可与12周龄人胎脑组织细胞的Ⅱ型角蛋白(K2C1)、Ⅰ型角蛋白(K1C10)、组蛋白H2B以及波形蛋白相互作用,但其相互作用的生物学意义及其在HCMV感染致胚胎CNS畸形发育中的作用有待进一步研究。
Objective: Proteins interacting with IE_286 from the brain tissue of 12-week-old embryo were screened by Pull-Down assay, to investigate the role of IE_286 in CNS anomalies induced by HCMV congenital infection and its possible mechanisms.
Methods: (1) UL122 CDS encoding IE_286 was amplified by PCR with pBT-IE_286 plasmid as template, and then the amplification product was digested with restriction endonucleases BamHI and NotI, and cloned into the corresponding sites on bait plasmid pGEX-4T-1. The recombinant was transformed into E. coli BL21. Followed by PCR, restriction and DNA sequence analysis. (2) GST-IE_286 fusion protein was expressed in 30℃for 3-4 hours under 1mM IPTG induction and purified by affinity column chromatography; GST protein was expressed and purified as negative control. (3) Fetal brain tissue from 12-week-old embryo was taking out and grinding into homogenate. The proteins interacting with IE_286 were screened from lysates by Pull-Down assay. (4) Cut the stably expressive protein bands and analysed the proteins with Quatropde-Time of Flight Mass spectrometer.
Results: (1) The pGEX-4T-1/IE_286 bait plasmid was constructed successfully. The result of DNA sequence analysis was no mutation compared with the sequence in GenBank. (2) GST- IE_286 fusion protein and GST protein were expressed and purified successfully. The protein molecular weight of GST- IE_286 fusion protein was 92kDa, and GST protein was 26kDa, consistent with the expected protein molecular weight. (3) The fetal brain protein which involved with IE_286 has been found by using the method of Pull-Down assay, after mass spectrometry detection, there are four kinds of protein: Keratin, type II cytoskeletal 1, Keratin, type I cytoskeletal 10, Histone, H2B type 1-B and Vimentin.
Conclusions: HCMV IE_286 may have interaction with four kinds of protein from 12-week-old embryo: Keratin, type II cytoskeletal 1, Keratin, type I cytoskeletal 10, Histone, H2B type 1-B and Vimentin, however, the biological significance and the role of these interaction in fetal congenital anomalies of the central nervous system (CNS) which caused by infection of HCMV need to be further studied.
引文
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