狂犬病病毒Evelyn- Rokitnicki-Abelseth疫苗株反向遗传系统的建立

摘要：【目的】建立狂犬病毒的反向遗传系统,为研制不含狂犬病病毒致病性的新型安全高效的狂犬疫苗提供技术依据。【方法】本研究采用反向遗传学方法和分子克隆技术,建立了狂犬病病毒Evelyn- Rokitnicki-Abelseth (ERA)疫苗株的CMV/ T7、T7启动子病毒拯救系统,构建了表达N、P、L蛋白的辅助质粒。【结果】成功拯救出野生型病毒rERA-VC,在Vero细胞上的生长动力学特性与父母本ERA 相同,第三代可在Vero细胞上可获得很高的生长滴度。【结论】建立了狂犬病病毒的反向遗传系统,拯救出的野生型病毒生物学特性与父母本相同。     

狂犬病病毒EveIyn-Rokitnicki-Abelseth疫苗株反向遗传系统的建立

[目的]建立狂犬病毒的反向遗传系统,为研制不含狂犬病病毒致病性的新型安全高效的狂犬疫苗提供技术依据.[方法]本研究采用反向遗传学方法和分子克隆技术,建立了狂犬病病毒Evelyn-R0kitnieki-Abelseth(ERA)疫苗株的cMV/T7、T7启动子病毒拯救系统,构建了表达N、P、L蛋白的辅助质粒.[结果]成功拯救出野生型病毒rERA-VC,在Vero细胞上的生长动力学特性与父母本ERA相同,第三代可在Vero细胞上可获得很高的生长滴度.[结论]建立了狂犬病病毒的反向遗传系统,拯救出的野生型病毒生物学特性与父母本相同.     

美、英、比三国建成“抗虫工程植物”取得新进展

“抗虫工程植物”组建成功是农业生物工程研究的重要突破,也是植物分子遗传学研究的重要成果,一直引起世界生命科学工作者的密切关注,这三个国家在探究细菌毒素蛋白基因引入植物方面已取得突破,为组建“抗虫工程植物”开辟了一条新途径,并为“杀虫工程植物”早日实用化的实现而努力。     

反向遗传学技术在疫苗研究的进展

本文通过反向遗传学技术的基本原理,发展历史和反向遗传学技术在疫苗研究方面的进展与应用,重点介绍了反向遗传学技术在流感病毒疫苗方面的应用。     

案例在遗传与优生教学中的应用

“遗传与优生”课程是遗传学与优生学相结合的一门交叉性学科, 是国内许多高校在教育教学改革中开设的一门通识课程。通过案例进行教学可以有效提升学生学习的积极性, 收到较好的教学效果。文章针对“遗传与优生”课程教学中应用案例进行教学所应注意的问题进行了讨论。     

重视经典遗传学知识体系构建和学生自学能力的培养

遗传学知识的不断更新以及信息量快速增加, 这对遗传学的本科教学是一种挑战。作者认为, 在本科教学中应加强学生遗传学知识体系的构建, 即“把书读薄”, 同时注重提高学生主动学习的能力, 即“把书读厚”, 以提高学生自学能力和逻辑思维能力, 为培养具有一定竞争力的复合型人才奠定坚实的基础。     

“表观遗传学研究进展专刊”编者寄语

近年来, 表观遗传学和表观基因组学已经成为生命科学领域的研究热点。为了向广大读者展示国内外最新研究成果与进展, 2012年12月1日在昆明召开的“八届三次《遗传》编委会议”上决定组织出版“表观遗传学研究进展专刊”, 由朱卫国、宋旭、张根发、李绍武等编委负责组稿约稿。大家积极工作, 共组稿30余篇, 经正常程序送审后, 共录用20篇, 定于2014年第3期、第5期分两次出版。     

反向遗传学在现代生物学领域中的应用

反向遗传学是一种新兴的分子生物学技术.主要从反向遗传学的含义、研究方法及应用等角度进行综述,并介绍有关反向遗传学研究的最新进展.     

基因工程的现状与未来

今天,人们听到“生物工艺学”、“遗传工程”和“重组DNA”这些名词的机会,明显地多起来了。这方面的研究,已经向实际应用方面发展。看来这些技术已经引起各阶层人士的注意和兴趣。但是,人们又对这突然出现的热潮感到茫然。     

食品加工企业申请使用绿色食品品牌的影响因素及对策

通过实证分析的方法,利用1 900多家企业问卷调查样本,分析食品加工企业申请使用绿色食品品牌的影响因素,建立了从企业视角衡量绿色食品品牌发展的研究思路,设置了“是否发展”、“是否续展”、“是否用标”3个被解释变量,研究了绿色食品品牌的作用、外部政策因素、企业顾虑的因素以及企业认知等因素对企业决策的影响。分析得出,绿色食品的品牌提升和质量保障作用对企业的激励较大,而面临的监管压力对企业发展绿色食品、续展和在包装上用标均有反向影响。     

Construction and identification of the helper plasmids for reverse genetic system of rabies virus street strain

To obtain the helper plasmids for a reverse genetics system of rabies virus, the cDNAs of the complete open reading frames of the N, P, G, and L genes of rabies street virus stain HN10 were each cloned into expression vector pVAX1, These four plasmids were identified by restriction enzyme digestion and gene sequencing. The plasmid encoding the N protein was selected to determine the expression effect of these plasmids in NA cells. The results showed that the helper plasmids for a reverse genetics system of rabies street virus strain HN10 had been successfully constructed.     

Characterization of M gene-deficient rabies virus with advantages of effective immunization and safety as a vaccine strain

Matrix (M) protein of rabies virus is known to play an important role in assembly and budding of the progeny virus. We generated an M gene-deficient rabies virus, RC-HLDeltaM, using a reverse genetics system of rabies virus RC-HL strain to develop a novel type of vaccine. RC-HLDeltaM infection was confined within a single cell in mouse neuroblastoma cells. This deficient virus failed to generate the progeny virus in the cells. In contrast, RC-HLDeltaM propagated in BHK cells inductively expressing M protein. Suckling and adult mice inoculated intracerebrally with the parental RC-HL strain showed lethal infection and transient body weight loss, respectively, whereas both suckling and adult mice inoculated with RC-HLDeltaM showed no symptoms. The neutralizing antibody against rabies virus was successfully induced by intramuscular immunization with 10(5) focus-forming units of RC-HLDeltaM but not UV-inactivated RC-HL. Intranasal immunization with RC-HLDeltaM resulted in almost the same antibody titer to rabies virus as that in the case of immunization with live RC-HL strain. These findings indicate that RC-HLDeltaM is a candidate for a novel rabies vaccine that is safer and more effective than are current vaccines.     

用lac基因筛选表达狂犬病毒糖蛋白的重组痘苗病毒

为了研制基因工程狂犬病疫苗,我国于1991年首次报道了在痘苗病毒天坛株中表达狂犬病毒糖蛋白,但报道中重组病毒的选择是先经人骨髓瘤细胞(TK-143)在诱变剂5-溴脱氧尿苷(BrudR)作用下通过标记拯救技术筛选出携带有同源基因的重组病毒,然后再利用重组病毒中携带的Lac基因为选择标记,通过噬斑纯化获得重组病毒,用这种选择方式获得的重组病毒,经过了TK-143细胞和BrudR,因此不宜发展成疫苗,本研究探索不经过TK-143细胞和BrudR,仅利用Lac基因为选择标记,直接在鸡胚细胞上通过噬斑纯化获得重组病毒,现将研究结果报道如下。     

Genetic analysis of rabies virus isolates in the Philippines

To determine the genetic characteristics of the rabies virus in the Philippines, 59 rabies virus isolates were obtained from domestic rabid dogs and their partial nucleotide sequences of nucleoprotein (N) gene were compared. Based on comparison with reported sequences, phylogenetic analysis revealed that all isolates from the Philippines had close genetic relations and formed two subgroups. The Philippines isolates belonged to a different lineage from other Asian isolates but were closer to them than to terrestrial isolates and laboratory strains. Several specific nucleotide and amino acid substitutions were observed among the Philippines isolates. Our results suggest that rabies viruses in the Philippines might have a characteristic evolution.     

糖蛋白G 基因在基因组P-M 位的插入重组表达对狂犬病毒Flury LEP 致病力的影响

【目的】研究狂犬病病毒Flury鸡胚低代毒株(Flury LEP)在基因组P-M位增加糖蛋白基因(G基因)的重组表达对病毒致病力的影响。【方法】利用反向遗传操作技术,构建了P、M基因之间额外插入G基因的重组狂犬病病毒Flury LEP株(rLEP-PGM),并对重组病毒的生物学特性及对小鼠的致病性进行了初步研究。【结果】亲本株和重组病毒具有相似的生长特性,LEP和rLEP-PGM在BHK-21细胞的生长滴度分别为4×106 FFU/mL和2.5×106 FFU/mL,在小鼠神经母细胞(NA)的生长滴度分别为4×107 FFU/mL和2.5×107 FFU/mL;嗜神经指数均为1;Western blot显示,rLEP-PGM在感染细胞的G蛋白表达量比LEP显著提高;小鼠感染试验显示,rLEP-PGM与LEP脑内注射小鼠的LD50分别为3 FFU和1 FFU,肌肉注射途径的LD50分别为4×104 FFU和3.2×105 FFU。【结论】P、M基因之间插入一个额外的G基因能够提高G蛋白的表达水平,同时增强重组病毒外周侵入中枢神经系统的能力。     

Target Cells of Cytotoxic T Lymphocytes Directed to the Individual Structural Proteins of Rabies Virus

Target cells of cytotoxic T lymphocytes (CTL) directed to the individual structural proteins (except for the large polymerase (L) protein) of rabies virus were established by expressing only the respective protein in murine neuroblastoma (NA) and murine macrophage (J774-1) cell lines. Mice infected with the ERA strain of rabies virus developed CTL responses to all of these rabies virus proteins. The cytotoxic activity was abrogated by pretreatment of the effector cells with anti-CD8 monoclonal antibody (MAb) and complement but not with anti-CD4 MAb. Cell lysis by CTL was blocked in the presence of anti-major histocompatibility complex (MHC) class 1 antibodies in J774-1 cell lines. Rabies virus-infected cells express these proteins at the surface, which can be recognized and lysed by the respective CTL. Mice immunized with β-propiolactone-inactivated virus induced a CTL response against glycoprotein but not against internal viral components. This assay system might be useful for further analysis of the possible contribution of these proteins in the cell-mediated immune protection against rabies.