目的 应用酵母双杂交技术筛选小鼠脑cDNA文库中与鼠巨细胞病毒即刻早期蛋白M122相互作用的宿主因子,为进一步研究巨细胞病毒的致病机制奠定实验基础.方法将诱饵质粒pGBKT7-M122转化酵母菌AH109,Western blot检测诱饵蛋白在酵母细胞中的表达.阳性重组AH109菌株与小鼠脑cDNA文库进行配合,在色氨酸、亮氨酸、组氨酸和腺嘌呤缺陷培养基(SD/-Trp/-Leu/-His/-Ade)和铺有Ⅹ-α-gal的SD/-Trp/-Leu/-His/-Ade平板上进行筛选,提取阳性酵母菌质粒,转化大肠杆菌后提取质粒测序,对测序结果进行序列比对和分析.将阳性文库质粒与诱饵质粒共同转化酵母AH109感受态细胞,重新验证其在酵母中的相互作用,同时阳性文库质粒与空载体pGBKT7亦被用同样的方法转入AH109感受态细胞,以排除阳性文库质粒的自激活作用.结果筛选出与M122蛋白相互作用的21种已知基因编码的蛋白质和3种未知基因编码的蛋白,其中21种已知蛋白分别为:突触融合蛋白8(syntaxin 8,Stx8)、磷酸葡萄糖变位酶2(phosphoglucomutase 2,Pgm2)、Shaker型电压依赖性钾通道的β1亚单位(potassium voltage-gated channel,shaker-related subfamily,beta member 1,Kcnab1)、19型胶原蛋白(collagen,type ⅪⅩ,alpha 1,Col19a1)、古蛋白1(archain 1,Arcn1)、胞嘧啶核苷酸激酶(cytidylate kinase,Cmpk)、热休克蛋白DnaJ同系物A亚家族成员1[DnaJ(Hsp40)homolog,subfamily A,member 1,Dnaja1]、Na+、K+ATP转运酶β3亚单位(ATPase,Na+/K+ transporting,beta 3 polypeptide,Atp1b3)、SH3结构域GRB2样相互作用蛋白1[SH3-domain GRB2-like(endophilin)interacting protein 1,Sgip1]、锚蛋白重复域17(ankyrin repeat domain 17,Ankrd17)、无义介导的mRNA 降解因子Smg-7同系物(Smg-7 homolog,nonsense mediated mRNA decay factor,Smg7)、精子相关抗原9(sperm associated antigen 9,Spag9)、FK506结合蛋白1A(FK506 binding protein 1a,Fkbp1a)、MYST组蛋白乙酰转移酶4[MYST histone acetyltransferase(monocytic leukemia)4,Myst4]、透明质酸和蛋白多糖连接蛋白1(hyaluronan and proteoglycan link protein 1,Hapln1)、自噬相关蛋白3(autophagy-related 3,Atg3)、精氨酸/色氨酸富集的剪切因子5(splicing factor,arginine/serine-rich 5,Sfrs5)、C3HC型锌指蛋白(zinc finger,C3HC-type containing 1,Zc3hc1)、硫氧还蛋白相关的跨膜蛋白1(thioredoxin-related transmembrane protein 1,Txndc1)、接头蛋白复合物AP-1亚单位(adaptor protein complex AP-1,gamma 1 subunit,Aplg1)和Cul1蛋白(cullin 1,Cul1).回返验证实验进一步证实这些蛋白与M122蛋白能够在酵母细胞AH109发生相互作用,但Aplg1和Cul1被证实具有自激活作用.结论 筛选到的其中19种已知基因编码的蛋白可能与巨细胞病毒的致病机制相关,但仍需进一步的验证.
目的 應用酵母雙雜交技術篩選小鼠腦cDNA文庫中與鼠巨細胞病毒即刻早期蛋白M122相互作用的宿主因子,為進一步研究巨細胞病毒的緻病機製奠定實驗基礎.方法將誘餌質粒pGBKT7-M122轉化酵母菌AH109,Western blot檢測誘餌蛋白在酵母細胞中的錶達.暘性重組AH109菌株與小鼠腦cDNA文庫進行配閤,在色氨痠、亮氨痠、組氨痠和腺嘌呤缺陷培養基(SD/-Trp/-Leu/-His/-Ade)和鋪有Ⅹ-α-gal的SD/-Trp/-Leu/-His/-Ade平闆上進行篩選,提取暘性酵母菌質粒,轉化大腸桿菌後提取質粒測序,對測序結果進行序列比對和分析.將暘性文庫質粒與誘餌質粒共同轉化酵母AH109感受態細胞,重新驗證其在酵母中的相互作用,同時暘性文庫質粒與空載體pGBKT7亦被用同樣的方法轉入AH109感受態細胞,以排除暘性文庫質粒的自激活作用.結果篩選齣與M122蛋白相互作用的21種已知基因編碼的蛋白質和3種未知基因編碼的蛋白,其中21種已知蛋白分彆為:突觸融閤蛋白8(syntaxin 8,Stx8)、燐痠葡萄糖變位酶2(phosphoglucomutase 2,Pgm2)、Shaker型電壓依賴性鉀通道的β1亞單位(potassium voltage-gated channel,shaker-related subfamily,beta member 1,Kcnab1)、19型膠原蛋白(collagen,type ⅪⅩ,alpha 1,Col19a1)、古蛋白1(archain 1,Arcn1)、胞嘧啶覈苷痠激酶(cytidylate kinase,Cmpk)、熱休剋蛋白DnaJ同繫物A亞傢族成員1[DnaJ(Hsp40)homolog,subfamily A,member 1,Dnaja1]、Na+、K+ATP轉運酶β3亞單位(ATPase,Na+/K+ transporting,beta 3 polypeptide,Atp1b3)、SH3結構域GRB2樣相互作用蛋白1[SH3-domain GRB2-like(endophilin)interacting protein 1,Sgip1]、錨蛋白重複域17(ankyrin repeat domain 17,Ankrd17)、無義介導的mRNA 降解因子Smg-7同繫物(Smg-7 homolog,nonsense mediated mRNA decay factor,Smg7)、精子相關抗原9(sperm associated antigen 9,Spag9)、FK506結閤蛋白1A(FK506 binding protein 1a,Fkbp1a)、MYST組蛋白乙酰轉移酶4[MYST histone acetyltransferase(monocytic leukemia)4,Myst4]、透明質痠和蛋白多糖連接蛋白1(hyaluronan and proteoglycan link protein 1,Hapln1)、自噬相關蛋白3(autophagy-related 3,Atg3)、精氨痠/色氨痠富集的剪切因子5(splicing factor,arginine/serine-rich 5,Sfrs5)、C3HC型鋅指蛋白(zinc finger,C3HC-type containing 1,Zc3hc1)、硫氧還蛋白相關的跨膜蛋白1(thioredoxin-related transmembrane protein 1,Txndc1)、接頭蛋白複閤物AP-1亞單位(adaptor protein complex AP-1,gamma 1 subunit,Aplg1)和Cul1蛋白(cullin 1,Cul1).迴返驗證實驗進一步證實這些蛋白與M122蛋白能夠在酵母細胞AH109髮生相互作用,但Aplg1和Cul1被證實具有自激活作用.結論 篩選到的其中19種已知基因編碼的蛋白可能與巨細胞病毒的緻病機製相關,但仍需進一步的驗證.
목적 응용효모쌍잡교기술사선소서뇌cDNA문고중여서거세포병독즉각조기단백M122상호작용적숙주인자,위진일보연구거세포병독적치병궤제전정실험기출.방법장유이질립pGBKT7-M122전화효모균AH109,Western blot검측유이단백재효모세포중적표체.양성중조AH109균주여소서뇌cDNA문고진행배합,재색안산、량안산、조안산화선표령결함배양기(SD/-Trp/-Leu/-His/-Ade)화포유Ⅹ-α-gal적SD/-Trp/-Leu/-His/-Ade평판상진행사선,제취양성효모균질립,전화대장간균후제취질립측서,대측서결과진행서렬비대화분석.장양성문고질립여유이질립공동전화효모AH109감수태세포,중신험증기재효모중적상호작용,동시양성문고질립여공재체pGBKT7역피용동양적방법전입AH109감수태세포,이배제양성문고질립적자격활작용.결과사선출여M122단백상호작용적21충이지기인편마적단백질화3충미지기인편마적단백,기중21충이지단백분별위:돌촉융합단백8(syntaxin 8,Stx8)、린산포도당변위매2(phosphoglucomutase 2,Pgm2)、Shaker형전압의뢰성갑통도적β1아단위(potassium voltage-gated channel,shaker-related subfamily,beta member 1,Kcnab1)、19형효원단백(collagen,type ⅪⅩ,alpha 1,Col19a1)、고단백1(archain 1,Arcn1)、포밀정핵감산격매(cytidylate kinase,Cmpk)、열휴극단백DnaJ동계물A아가족성원1[DnaJ(Hsp40)homolog,subfamily A,member 1,Dnaja1]、Na+、K+ATP전운매β3아단위(ATPase,Na+/K+ transporting,beta 3 polypeptide,Atp1b3)、SH3결구역GRB2양상호작용단백1[SH3-domain GRB2-like(endophilin)interacting protein 1,Sgip1]、묘단백중복역17(ankyrin repeat domain 17,Ankrd17)、무의개도적mRNA 강해인자Smg-7동계물(Smg-7 homolog,nonsense mediated mRNA decay factor,Smg7)、정자상관항원9(sperm associated antigen 9,Spag9)、FK506결합단백1A(FK506 binding protein 1a,Fkbp1a)、MYST조단백을선전이매4[MYST histone acetyltransferase(monocytic leukemia)4,Myst4]、투명질산화단백다당련접단백1(hyaluronan and proteoglycan link protein 1,Hapln1)、자서상관단백3(autophagy-related 3,Atg3)、정안산/색안산부집적전절인자5(splicing factor,arginine/serine-rich 5,Sfrs5)、C3HC형자지단백(zinc finger,C3HC-type containing 1,Zc3hc1)、류양환단백상관적과막단백1(thioredoxin-related transmembrane protein 1,Txndc1)、접두단백복합물AP-1아단위(adaptor protein complex AP-1,gamma 1 subunit,Aplg1)화Cul1단백(cullin 1,Cul1).회반험증실험진일보증실저사단백여M122단백능구재효모세포AH109발생상호작용,단Aplg1화Cul1피증실구유자격활작용.결론 사선도적기중19충이지기인편마적단백가능여거세포병독적치병궤제상관,단잉수진일보적험증.
Objective To investigate the biological function of M122 in pathogenesis of MCMV in developmental brain disorders and brain damage, screening for mouse brain cDNA library interacting with M122 was performed by a yeast two-hybrid system. Methods The reconstructed bait plasmid pGBKT7-M122 was transformed into yeast cells AH109 and screened on the nutrient deficiency medium SD/-Trp. After express of the bait protein in AH109 yeast strains was detected by Western blot analysis, yeast-two hybrid screening was performed by mating AH109 with Y187 containing mouse brain cDNA library plasmid. The diploid yeast cells were plated on the nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade. The second screening was performed with SD/-Trp/-Leu/-His/-Ade containing X-α-gal. The plasmids in positive colonies were extracted and transformed into E. coli JM109 cells. After plasmid DNA in JM109 cells were extracted form positive colonies and sequenced, the results were analyzed by bioinformatic methods. The interactions between M122 protein and the protein obtained from positive colonies were further confirmed by repeating yeast-two hybrid. Then, autoactivations of the proteins obtained from positive colonies were detected.Results The reconstructed bait plasmid was transformed into yeast cells AH109 successfully. The bait protein expressed in the yeast cells AH109 stably. 24 proteins interacting with MCMV M122 were screened, including syntaxin 8 ( Stx8 ), phosphoglucomutase 2 ( Pgm2 ), potassium voltage-gated channel, shaker-related subfamily, beta member 1 ( Kcnab1 ), collagen, type ⅪⅩ, alpha 1 ( Col19a1 ), archain 1 ( Arcn1 ), cytidylate kinase( Cmpk), DnaJ(Hsp40) homolog, subfamily A, member 1 (Dnaja1), ATPase, Na+/K + transporting, beta 3 polypeptide( Atp1b3 ), SH3-domain GRB2-like ( endophilin ) interacting protein 1 ( Sgip1 ),ankyrin repeat domain 17 (Ankrd17), Smg-7 homolog, nonsense mediated mRNA decay factor(Smg7),sperm associated antigen 9 ( Spag9 ), FK506 binding protein 1a ( Fkbp1a), MYST histone acetyltransferase monocytic leukemia 4 ( Myst4), hyaluronan and proteoglycan link protein 1 ( Hapln1), autophagy-related 3 (Atg3), splicing factor, arginine/serine-rich 5 ( Sfrs5 ), zinc finger, C3HC-type containing 1 ( Zc3hc1 ),thioredoxin-related transmembrane protein 1 ( Txndc1 ), adaptor protein complex AP-1, gamma 1 subunit (Ap1g1), Cullin 1 ( Cul1 ), and so on. Three of them were formerly unknown proteins. M122 protein could interact with the proteins obtained from positive colonies in the yeast cells AH109. Ap1g1 and Cul1 were proved to have autoactivation. Conclusion A class of proteins in brain interacting with M122 has been obtained. It is presumed that these proteins are correlated with neuropathogenesis of the brain disorders caused by CMV, but the candidates still need further confirmation for the interaction.
