表达O型口蹄疫病毒 VP1基因的重组病毒BHV-1的构建与鉴定

摘要：【目的】为了构建表达口蹄疫病毒（O/China/99）VP1基因的牛疱疹病毒1型,将人工合成的口蹄疫病毒VP1基因插入到巨细胞病毒（CMV）启动子之下构建gE基因缺失转移载体。【方法】利用磷酸钙介导转染法将该转移载体与亲本病毒BHV-1/gE-/LacZ+的基因组DNA共转染牛鼻甲细胞后收获增殖的病毒。通过筛选白色病毒蚀斑,得到重组病毒BHV-1/gE-/VP1。【结果】PCR检测结果表明VP1基因已经插入到了重组病毒BHV-1/gE-的基因组中,间接免疫荧光试验和Western blot证实了BHV-1/gE-/VP1中的VP1基因在感染的细胞中获得了表达。【结论】本研究成功的构建了表达口蹄疫病毒VP1基因的重组病毒BHV-1/gE-/VP1,为研制口蹄疫及其他重要牛传染病的BHV-1病毒载体疫苗奠定了基础。     

表达猪细小病毒VP2基因的重组伪狂犬病毒的构建及其生物学特征研究

根据Bergeron等报道的猪细小病毒(PPV)基因组,设计一对包含VP2全基因的PCR引物,上下游均引入一个BamHI位点,扩增得到VP2基因后,将其插入到pUSK载体中,构建了转移载体pUSK-VP2.采用脂质体介导的转染方法,将伪狂犬病毒TK-/gG-/LacZ 株的基因组DNA与pUSK-VP2共转染PK-15细胞,待细胞病变后收集病毒液,在空斑纯化的同时,利用检测PPV VP2基因和LacZ基因的PCR方法筛选重组病毒TK-/gG-/VP 2株,Southern blotting、SDS-PAGE、Western blotting和电镜观察鉴定重组病毒,并在不同细胞上测定重组病毒的增殖滴度,接种小鼠进行安全性试验.结果发现,外源基因VP2已成功地插入到TK-/gG-/LacZ 亲本株的基因组中,并获得了表达.表达的VP2蛋白可以与猪细小病毒阳性血清反应,而且可以自行装配成病毒样颗粒.同时发现,VP2基因的插入不影响重组病毒的增殖特性,其毒力与亲本株相当.     

转录病毒载体介导的稳定表达口蹄疫病毒3D基因的BHK21细胞系的建立

[目的]研究口蹄疫病毒RNA聚合酶在BHK-21细胞中的稳定表达状况,为研究RNA聚合酶生物学活性及其基囚工程疫苗研制提供科学依据.[方法]从重组质粒pMD18-T-3D扩增口蹄疫病毒3D基因,通过分子克隆技术构建莺组逆转录病毒表达载体pBPSTR1-3D.用pBPSTR1-3D和pVSV-G双质粒瞬时转染GP2-293包装细胞,收获重组逆转录病毒,然后感染BHK-21细胞,嘌呤霉素持续筛选12 d后获得阳性克隆,并用有限稀释法挑选单个阳性细胞克隆.[结果]应用PCR、RT-PCR技术可从体外反复传代的阳性细胞中扩增到3D基因,证实目的外源基因能转录并被稳定整合进宿主细胞基因组中.经SDS-PAGE、Western blot、间接免疫荧光检测到在不同代次的阳性细胞中有目的蛋白表达.[结论]本试验利用逆转录病毒载体介导的基因转移技术,将外源基因插入到靶细胞的基因组中,构建了稳定表达口蹄疫病毒RNA聚合酶的包装细胞系,为研究3D基因表达及其蛋白定位提供了方便,也为下一步研究RNA聚合酶生物学功能和疫苗研制提供了科学依据.     

共表达口蹄疫病毒衣壳前体蛋白P1-2A基因和蛋白酶3C基因牛肾细胞株的筛选及稳定性

[目的]筛选稳定表达口蹄疫病毒衣壳蛋白的牛肾细胞(Madin-Darby bovinekidney,MDBK)株.[方法]采用聚合酶链式反应(Polymerase chain reaction,PCR)方法从重组质粒pMD-P1-2A和pMD-3C中分别扩增口蹄疫病毒衣壳前体蛋白P1-2A基因和蛋白酶3C基因,将两基因依次插入逆转录病毒载体pBABE-puro.重组逆转录病毒载体pBABE-puro/P1-2A-3C和pVSV-G质粒载体用脂质体介导共转染GP2-293包装细胞.产生的重组逆转录病毒感染MDBK细胞后使用嘌呤霉素筛选抗性细胞.利用克隆环套取法得到单克隆细胞.经间接免疫荧光和酶联免疫吸附测定(Enzyme-linkedimmunosorbent assay,ELISA)方法检测MDBK细胞中衣壳蛋白的表达,并在电镜下观察口蹄疫病毒空衣壳.[结果]成功筛选到稳定表达口蹄疫病毒衣壳蛋白的MDBK细胞株,衣壳前体蛋白P1-2A在蛋白酶3C裂解作用下正确组装成空衣壳.[结论]该研究为口蹄疫亚单位疫苗的研制提供了实验材料.     

Ⅰ型牛疱疹病毒TK-/gE-基因双缺失突变株生物学特性

目的构建Ⅰ型牛疱疹病毒（BHV-1）的TK-/gE-双缺失突变株,检测其生物学功能,为治疗牛传染性鼻气管炎提供参考。方法构建了带有EGFP表达盒的BHV-1 TK-/gE-双缺失突变株。通过southern杂交、蛋白质斑点印迹、空斑试验、MDBK细胞增殖实验等技术方法,对重组基因所构成病毒突变株的生物学特性进行鉴定。结果 Southern杂交及蛋白斑点印迹表明,课题组成功构建了带有EGFP表达盒的双缺失突变株;该突变株具有与野生株相当的繁殖力,但TK-/gE-成功缺失,毒力大幅减弱;BHV-1 TK-/gE-遗传稳定,不会返强。结论本项目组成功构建了Ⅰ型牛疱疹病毒（BHV-1）的TK-/gE-双缺失突变株,该缺失株具有良好的安全性和稳定性,为该突变株进一步作为生物安全疫苗和多价病毒载体提供了依据,为预防和治疗牛传染性鼻气管炎提供了技术支持。     

表达绿色荧光蛋白报告基因重组禽腺联病毒载体系统的构建与鉴定

为了开展重组禽腺联病毒(Recombinant avian adeno-associated virus,rAAAV)介导的基因转移研究,用限制性内切酶消化含AAAV全基因组的重组质粒pCR-AAAV,去除AAAV Rep和Cap蛋白编码序列,将PCR扩增的绿色荧光蛋白(Green fluorescent protein,GFP)报告基因插入AAAV末端反向重复序列(Inverted terminal repeats,ITR)之间,获得表达GFP基因的AAAV转移载体pAITR-GFP;以含AAAV全基因组的质粒为模板,用PCR分别扩增AAAV Rep、Cap和Rep-Cap蛋白基因,将Rep和Cap基因分别插入真核细胞双表达载体pVITRO2-mcs的两个多克隆位点,将Rep-Cap蛋白基因插入真核细胞表达载体pcDNA3,获得AAAV辅助质粒pVITRO2-ARC和pcDNA-ARC;将pAITR-GFP、pVITRO2-ARC或pcDNA-ARC与腺病毒辅助质粒pHelper组成三质粒转染系统,用磷酸钙沉淀法共转染AAV-293细胞,获得了表达GFP的rAAAV.经SDS-聚丙烯酰胺凝胶电泳分离后,纯化病毒出现分子量正确的VP1、VP2、VP3结构蛋白;经PCR检测证明,重组病毒中含有GFP报告基因;用重组病毒分别感染鸡胚成纤维细胞(CEF)和鸡胚肝CEL细胞,可以观察到GFP报告基因的表达,表达时间持续两周以上.这些试验结果表明,成功建立了辅助病毒非依赖性rAAAV体外包装体系,为禽源细胞的基因转移研究和禽重组活载体疫苗的研制打下了基础.     

鸭病毒性肝炎病毒VP1基因表达及其抗体检测ELISA方法的建立

[目的]研究DHV VP1基因在大肠杆菌中的表达,并以纯化的重组蛋白为抗原建立鸭病毒性肝炎抗体检测ELISA方法.[方法]采用RT-PCR技术,扩增VP1基因,与Pmd18-T载体进行连接,构建DHV VP1基因克隆重组质粒.然后定向插入到Pet-32a( )表达载体,筛选原核表达载体Pet-32a-VP1,进行ITPG诱导表达分析.以纯化的重组蛋白为抗原建立间接ELISA方法并初步应用于临床.[结果]DHV VP1基因可在大肠杆菌中稳定、高效地表达.Western blot检测表明,表达的重组蛋白能与鸭肝炎阳性血清发生特异性反应.确定间接ELISA方法的抗原最佳包被浓度为5 ug/孔,血清最佳稀释度为1:100,临界值为OD450值≥0.302,建立的ELISA方法具有较好的敏感性、特异性和重复性.通过对80份血清样品的检测表明,该方法与中和试验的符合率为97.5%,初步临床应用结果表明该方法可用于雏鸭母源抗体和免疫后抗体的消长变化的检测.[结论]以大肠杆菌表达的DHV VP1重组蛋白为抗原建立的间接ELISA方法可用于鸭病毒性肝炎抗体的检测.     

含口蹄疫病毒VP1基因与结核杆菌HSP70基因的重组腺病毒转移载体的构建

根据GenBank已发表的结核杆菌HSP70的基因序列及O型口蹄疫病毒VP1基因序列,应甩Primer5．0软件设计了2对引物,其中一对引物用于扩增结核杆菌基因组中的HSP70完整基因,另一对引物用于以pMD-D为模板,扩增其中的VP1完整基因序列。将所扩增的2段基因串联插入腺病毒转移载体．pAdenovator-CMV5-IRES-GFP的BgⅢ位点,经PCR方法和限制性酶切方法鉴定2基因已成功插入了转移载体并且插入方向正确,获得了重组腺病毒转移载体pAdenovator-CMV5-IRES-GFP-VP1-HSP,此转移载体可与腺病毒骨架载体进行细菌内同源重组,以产生重组腺病毒载体。     

口蹄疫病毒结构蛋白VP1容忍外源标签的能力

【目的】利用口蹄疫病毒的反向遗传操作技术,构建含不同外源标签口蹄疫病毒的全长克隆,鉴定口蹄疫病毒结构蛋白VP1容忍不同外源标签的能力。【方法】通过融合PCR技术,在FMDV O/HN/93全长感染性克隆的VP1 G-H环分别引入V5、TC12、KT3、3FLAG外源标签,构建全长质粒。全长质粒经Not I线化后转染表达T7 RNA聚合酶的稳定细胞,拯救重组病毒。RT-PCR、序列测定、间接免疫荧光鉴定病毒,噬斑和一步生长曲线分析重组病毒的生物学特性。【结果】成功拯救到表达V5或KT3表位标签的重组病毒,未能拯救到表达TC12或3×FLAG的重组病毒。V5和KT3表位标签的插入均影响了口蹄疫病毒的复制能力。【结论】重组口蹄疫病毒的成功拯救为未来标记疫苗以及口蹄疫病毒作为表达载体等的研究奠定了基础。     

腺病毒介导犬干扰素-γ基因的表达及其体外抗犬细小病毒的作用

[目的]构建含犬干扰素-γ(c IFN-γ)基因的重组腺病毒,并在培养的犬肾细胞MDCK中分析其抗犬细小病毒的活性.[方法]首先将cIFN-γcNDA基因克隆到腺病毒穿梭质粒中,构建成含cIFN-γ基因的腺病毒穿梭质粒pShuttle3-cIFN-γ.利用特异的酶切位点,通过直接连接法将cIFN-γ表达盒插入到腺病毒基因组质粒pAdeno-X中,构建成含cIFN-γ基因的腺病毒基因组质粒pAd-cIFN-γ.pAd-cIFN-γ质粒经酶切线性化后转染人胚胎肾细胞HEK293T,在细胞中拯救出含有cIFN-γ基因的复制缺陷型重组腺病毒.然后用该重组腺病毒处理(感染)培养的犬肾细胞MDCK,再用犬细小病毒感染重组腺病毒处理的细胞,分析重组腺病毒在体外抗犬细小病毒的活性.[结果]通过连接法构建了含cIFN-γ基因的重组腺病毒,构建的重组腺病毒能够介导cIFN-γ在MDCK细胞中进行分泌表达.用含cIFN-γ基因的重组腺病毒处理MDCK细胞,可明显地抑制犬细小病毒在细胞中的增殖,表明构建的重组腺病毒具有明显的抗犬细小病毒的活性.[结论]构建了含cIFN-γ基因的重组腺病毒,并证明该重组病毒在体外具有明显的抗犬细小病毒的活性.     

DEC1 and MIC-1

Comment on: Qian Y, et al. Proc Natl Acad Sci USA 2012; 109:11300-5.     

Comparative CYP1A1 and CYP1B1 substrate and inhibitor profile of dietary flavonoids

CYP1A1 and CYP1B1 are two extrahepatic enzymes that have been implicated in carcinogenesis and cancer progression. Selective inhibition of CYP1A1 and CYP1B1 by dietary constituents, notably the class of flavonoids, is a widely accepted paradigm that supports the concept of dietary chemoprevention. In parallel, recent studies have documented the ability of CYP1 enzymes to selectively metabolize dietary flavonoids to conversion products that inhibit cancer cell proliferation. In the present study we have examined the inhibition of CYP1A1 and CYP1B1-catalyzed EROD activity by 14 different flavonoids containing methoxy- and hydroxyl-group substitutions as well as the metabolism of the monomethoxylated CYP1-flavonoid inhibitor acacetin and the poly-methoxylated flavone eupatorin-5-methyl ether by recombinant CYP1A1 and CYP1B1. The most potent inhibitors of CYP1-EROD activity were the methoxylated flavones acacetin, diosmetin, eupatorin and the di-hydroxylated flavone chrysin, indicating that the 4'-OCH(3) group at the B ring and the 5,7-dihydroxy motif at the A ring play a prominent role in EROD inhibition. Potent inhibition of CYP1B1 EROD activity was also obtained for the poly-hydroxylated flavonols quercetin and myricetin. HPLC metabolism of acacetin by CYP1A1 and CYP1B1 revealed the formation of the structurally similar flavone apigenin by demethylation at the 4'-position of the B ring, whereas the flavone eupatorin-5-methyl ether was metabolized to an as yet unidentified metabolite assigned E(5)M1. Eupatorin-5-methyl ether demonstrated a submicromolar IC(50) in the CYP1-expressing cancer cell line MDA-MB 468, while it was considerably inactive in the normal cell line MCF-10A. Homology modeling in conjunction with molecular docking calculations were employed in an effort to rationalize the activity of these flavonoids based on their CYP1-binding mode. Taken together the data suggest that dietary flavonoids exhibit three distinct modes of action with regard to cancer prevention, based on their hydroxyl and methoxy decoration: (1) inhibitors of CYP1 enzymatic activity, (2) CYP1 substrates and (3) substrates and inhibitors of CYP1 enzymes.     

Nucleic acid binding and unfolding properties of ribosomal protein S1 and the derivatives S1-F1 and m1-S1.

The nucleic acid binding and unwinding properties of wild-type Escherichia coli ribosomal protein S1 have been compared to those of a mutant form and a large trypsin-resistant fragment, both reported recently [J. Mol. Biol. 127, 41-45 (1979) and J. Biol. Chem. 254, 4309-4312 (1979). The mutant (m1-S1) contains 77% and the fragment (S1-F1) 66% of the polypeptide chain length (approximately 600 amino acid residues) of protein S1. The mutant is active in protein synthesis in vitro; the fragment, although retaining one or more of the functional domains of S1, is inactive in protein synthesis. We find that m1-S1 is is almost as effective as S1 in binding to poly(rU), phage MS2 RNA and simian virus 40 (SV40) DNA, and in unfolding poly(rU) and the helical structures present in MS2 RNA and phi X174 viral DNA. S1-F1, however, binds to poly(rU) and denatured SV40 DNA, but not to MS2 RNA. It unfolds neither poly(rU), nor the residual secondary structure of MS2 RNA or phi X174 viral DNA. Thus, there appears to be a correlation between the loss in ability of S1 to unwind RNA and the loss in its ability to function in protein synthesis.     

NPC1L1 and cholesterol transport

The polytopic transmembrane protein, Niemann-Pick C1-Like 1 (NPC1L1), is enriched in the apical membrane of small intestine absorptive enterocytes where it mediates extracellular sterol transport across the brush border membrane. It is essential for intestinal sterol absorption and is the molecular target of ezetimibe, a potent cholesterol absorption inhibitor that lowers blood cholesterol in humans. NPC1L1 is also highly expressed in human liver. The hepatic function of NPC1L1 may be to limit excessive biliary cholesterol loss. NPC1L1-dependent sterol uptake seems to be a clathrin-mediated endocytic process and is regulated by cellular cholesterol content. Recently, NPC1L1 inhibition has been shown to have beneficial effects on components of the metabolic syndrome, such as obesity, insulin resistance, and fatty liver, in addition to atherosclerosis.     

Expression Profile of CYP1A1 and CYP1B1 Enzymes in Colon and Bladder Tumors

Background

The cytochrome P450 CYP1A1 and CYP1B1 enzymes are involved in carcinogenesis via activation of pro-carcinogenic compounds to carcinogenic metabolites. CYP1A1 and CYP1B1 have shown elevated levels in human tumors as determined by qRT-PCR and immunohistochemical studies. However studies that have examined CYP1 expression by enzyme activity assays are limited.

Results

In the current study the expression of CYP1A1 and CYP1B1 was investigated in a panel of human tumors of bladder and colorectal origin by qRT-PCR and enzyme activity assays. The results demonstrated that 35% (7/20) of bladder tumors and 35% (7/20) of colon tumors overexpressed active CYP1 enzymes. CYP1B1 mRNA was overexpressed in 65% and 60% of bladder and colon tumors respectively, whereas CYP1A1 was overexpressed in 65% and 80% of bladder and colon tumors. Mean mRNA levels of CYP1B1 and CYP1A1 along with mean CYP1 activity were higher in bladder and colon tumors compared to normal tissues (p<0.05). Statistical analysis revealed CYP1 expression levels to be independent of TNM status. Moreover, incubation of tumor microsomal protein in 4 bladder and 3 colon samples with a CYP1B1 specific antibody revealed a large reduction (72.5 ± 5.5 % for bladder and 71.8 ± 7.2% for colon) in catalytic activity, indicating that the activity was mainly attributed to CYP1B1 expression.

Conclusions

The study reveals active CYP1 overexpression in human tumors and uncovers the potential use of CYP1 enzymes and mainly CYP1B1 as targets for cancer therapy.