干扰素-γ(IFN-γ)是免疫反应中重要的免疫调节因子,对多种免疫细胞具有激活作用.利用家蚕杆状病毒表达系统在家蚕中高效表达具有活性的人γ干扰素(hIFN-γ),首先根据家蚕密码子偏好性对hIFN-γ的编码基因进行优化合成,将优化合成后的序列克隆到杆状病毒转移载体pBR中,然后与orf1629基因缺失的失活拯救型杆状病毒BmBacmid共转染BmN细胞,获得重组病毒reBmBac-hIFN-γ,最后利用获得的重组病毒感染家蚕并收获表达产物.采用微量细胞病变抑制法,在Vero/VSV-GFP系统中检测到重组hIFN-γ 的效价达到(3.69±2.02)×106 IU/mL.此外,重组hIFN-γ可以抑制猪蓝耳病病毒(PRRSV)在Marc-145细胞中的增殖.上述结果为利用家蚕生物反应器高效生产具有活性的人γ干扰素奠定了试验基础.

Purified occlusion bodies (OBs) of Mythimna (formerly Pseudaletia ) unipuncta (the true armyworm) granulovirus Hawaiian strain (MyunGV-A) were observed, showing typical GV morphological characteristics under scanning and transmission electron microscopy (EM). The genome of MyunGV-A was completely sequenced and analysed. The genome is 176,677 bp in size, with a G+C content of 39.79%. It contains 183 open reading frames (ORFs) encoding 50 or more amino acids with minimal overlap. Comparison of MyunGV-A with TnGV, XcGV, and HearGV genomes revealed extensive sequence similarity and collinearity, and the four genomes contain the same nine homologous regions ( hrs ) with conserved structures and locations. Three unique genes, 12 baculovirus repeated ORF (bro), 2 helicase, and 3 enhancin genes, were identified. In particular, two repeated genes (ORF39 and 49) are present in the genome, in reverse and complementarily orientations. Twenty-four OB proteins were identified from the putative protein database of MyunGV-A. In addition, MyunGV-A belongs to the Betabaculovirus group and is most closely related to TnGV (99% amino acid identity) according to a phylogenetic tree based on the combined amino acid sequences of 38 core gene contents.

[目的]本研究旨在分析甜菜夜蛾Spodoptera exigua蛹卵巢细胞建立细胞系的整个过程,探究细胞由体内到体外培养过程中其基因表达在转录水平的变化,为昆虫体外培养模型的建立提供理论基础.[方法]利用Illumina Hiseq测序平台对甜菜夜蛾蛹卵巢细胞离体培养过程中各阶段的细胞分别进行转录组测序,对获得注释的差异表达基因及其相关信号通路进行分析;通过荧光定量PCR对部分细胞周期相关基因(cycd和cdk4)、调控基因(cdc20,apc1,skp2和mad1)、增殖相关分子标志物(mcm4和pcna)在甜菜夜蛾卵巢细胞离体培养过程中的转录进行验证.[结果]甜菜夜蛾蛹卵巢细胞离体培养过程包括5个阶段:解剖获得离体的卵巢组织,卵巢组织贴壁培养后游离出原代细胞,细胞转化重新具备增殖能力,成功首次传代,以及能够连续传代15代以上建立细胞系.上述5个阶段的细胞经转录组测序、数据组装后共获得46 796条unigenes序列,组装得到序列长度完整性好;转录本unigenes序列拼接长度分布合理,样本碱基Q30均在94％以上.通过KEGG数据库获得注释的unigenes有1 473条,参与细胞过程紧密相关的20条信号通路,其中有92条unigenes在细胞周期信号通路中获得注释.聚类分析表明,在体内处于快速发育状态的卵巢细胞与同样处于增殖状态的细胞系基因表达模式非常接近.原代细胞由短暂停滞生长至成簇细胞的转化关键期,筛选到差异表达基因619个,cdk4在离体培养期表达量显著降低,cycd在细胞转化关键期之后表达量显著升高,cdc20,apc1,skp2和mad1在卵巢组织和细胞系的表达量显著高于原代细胞、转化关键期细胞和首次传代细胞的.从原代细胞至传代后,cycd的表达显著升高8.7倍,显著高于mcm4和pcna的变化水平.[结论]甜菜夜蛾卵巢细胞离体培养过程中5个阶段的细胞转录组测序获得的序列质量符合数据分析的基本要求.筛选获得了甜菜夜蛾蛹卵巢细胞经离体培养过程中的差异表达基因.原代细胞逆转增殖可能与cdk4,cycd,skp2和mad1等细胞周期调控基因表达有关.另外,cycd可作为原代细胞具备传代能力的标志物.

The cell lines provided excellent tools to understand the mechanism of biological phenomenon at the cellular and molecular levels. The continuous development of new cell culture technology is both of interest for use in biochemical, immunology, and virological studies. The transformation of cells of the primary culture is a key procedure for insect cell line establishment but little is known about the molecular basis of these changes. Here, we found that the cell cycle progression of the cells of the primary culture was delayed or arrested in G2/M by fluorescence-activated cell sorting analysis. In this study, two subtractive cDNA libraries were constructed to screen for immortal-related genes of Spodoptera exigua (Lepidoptera: Noctuidae). Gene ontology and pathway analysis indicated that members of the oxidative phosphorylation, PI3K-Akt signaling pathway, and the ubiquitin proteasome pathway are involved in processes leading toward cell immortalization merit further investigation. Our findings suggest that tumor-related genes or target genes of these pathways may contribute to the transformation of primary cell through regulation of G2/M cell cycle progression.

Background HearMNPV, a nucleopolyhedrovirus (NPV), which infects the cotton bollworm, Helicoverpa armigera , comprises multiple rod-shaped nucleocapsids in virion(as detected by electron microscopy). HearMNPV shows a different host range compared with H. armigera single-nucleocapsid NPV (HearSNPV). To better understand HearMNPV, the HearMNPV genome was sequenced and analyzed. Methods The morphology of HearMNPV was observed by electron microscope. The qPCR was used to determine the replication kinetics of HearMNPV infectious for H. armigera in vivo . A random genomic library of HearMNPV was constructed according to the “partial filling-in” method, the sequence and organization of the HearMNPV genome was analyzed and compared with sequence data from other baculoviruses. Results Real time qPCR showed that HearMNPV DNA replication included a decreasing phase, latent phase, exponential phase, and a stationary phase during infection of H. armigera . The HearMNPV genome consists of 154,196 base pairs, with a G + C content of 40.07%. 162 putative ORFs were detected in the HearMNPV genome, which represented 90.16% of the genome. The remaining 9.84% constitute four homologous regions and other non-coding regions. The gene content and gene arrangement in HearMNPV were most similar to those of Mamestra configurata NPV-B (MacoNPV-B), but was different to HearSNPV. Comparison of the genome of HearMNPV and MacoNPV-B suggested that HearMNPV has a deletion of a 5.4-kb fragment containing five ORFs. In addition, HearMNPV orf66, bro genes, and hr s are different to the corresponding parts of the MacoNPV-B genome. Conclusions HearMNPV can replicate in vivo in H. armigera and in vitro , and is a new NPV isolate distinguished from HearSNPV. HearMNPV is most closely related to MacoNPV-B, but has a distinct genomic structure, content, and organization.

杆状病毒的进化研究对于丰富杆状病毒生物学,理解病毒多样性十分重要,本文基于杆状病毒全基因组信息,对杆状病毒基因顺序系统进化、基因含量系统进化、组分矢量法的进化研究方法进行了总结。

The polyhedron particles infected the cotton bollworm,is denominated as Helicoverpa armigera nucleopolyhedrovirus-B(HaNPV-B),and have irregular shape which diameter is about 2 μm by electron microscope(EM)scanning.HaNPV-B infected Pseudaletia separate well while Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus(HaSNPV)did not,which means the effective host range of HaNPV-B and HearSNPV are different.A genomic library of HaNPV-B was constructed by "partial filling-in" method,and the 6 035 bp fragment was assembled,which contained 6 putative ORFs.The analysis showed that HaNPV-B is different from the HaSNPV in gene content and structure level.Those results indicated that HaNPV-B and HaSNPV represent two separate species.It provides an insight into the interactions between host and baculovirus,also,it could help to study baculovirus /insect coevolution.

A genomic library of Helicoverpa armigera multiple nucleocapsid nucleopolyhedrovirus(HearMNPV)was constructed by"partial filling-in" method,the inserted sequence showed high identity to that the 38k gene was identified and acquired,its open reading frame(ORF)has 903 base pairs which encoded 300 amino acids.It has a late gene motif TTAAG and behaves similarly to all baculovirus late gene.The amino acid sequence analysis showed that the HearMNPV 38K protein has higher identities with Alphabaculovirus and has more closer relationship with Alphabaculovirus.The prediction of 38K Protein tertiary structure showed its structure has 95% identity with that of phosphatase,and it was related with nucleocapsid assembling.

本文从乳酸基因工程茵、载体选择标记和表达系统的构建方面介绍食品级乳酸茵表达系统,以及乳酸茵基因表达系统的应用前景.

[Objective] The aim was to clone the Helicoverpa armigera multiple nucleocapsid nucleopolyhedrovirus(HearMNPV) fp25 K gene in order to provide the basis for studying the evolutionary mechanism of baculovirus deeply.[Method] A plasmid genomic library of HearMNPV was constructed by partial filling-in method,the HearMNPV fp25 K gene was obtained by identifying and analyzing the sequence of the inserted segment.[Result] The reading frames of HearMNPV fp25 K gene was composed of 588 nucleotide acid,encoding 195 amino acids.It had a late gene motif ATAAG and behaved to all baculovirus late genes.HearMNPV fp25 K had 5α-helix and 8 β-sheet.HearMNPV had the highest homology with Mamestra configurata NPV,being 97.0% and had the lowest homology with Lymantria dispar multinucleocapsid NPV,being only 19.3%.The average similarity of amino acid of fp25 K homologous genes between HearMNPV and GroupⅡNPVs,GVs were 55.6%,69.4% and 29.3%,resp.,which could be conferred that the HearMNPV had a closer relationship with the Group II NPVs.[Conclusion] The research laid the foundation for the molecular evolution and the biological control of baculovirus.

A genomic library of Helicoverpa armigera multiple nucleocapsid nucleopolyhedrovirus(HearMNPV) is constructed by "partial filling-in" method,and the 24,553 bp fragment is assembled,which containes 26 putative ORFs.Gene parity comparison of HearMNPV fragment arrangement with that of Mamestra configurata Nucleopolyhedrovirus-B(MacoNPV-B) indicates that it is a 5 466 bp deletion in HearMNPV,which is high homologous with a cluster ORFs of Xestia c-nigrum granulovirus(XecnGV) from orf60 to orf65.The data suggests that there are recombination between HearMNPV and XecnGV.Also,it provides an insight into the gene flow that may be naturally occurring in field populations of baculoviruses.

In order to obtain the whole genome sequence of Helicoverpa armigera multiple nucleocapsid nucleopolyhedrovirus(HearMNPV),the genomic DNA of HearMNPV was partially digested by Sau3A I,and the plasmid vector pUC19 was fully digested by SalⅠ,and subsequently filled in by the Klenow fragment.After ligation and transformation,the inserted sequence showing high identity with that of the v-cath gene was acquired and identified.Its open reading frame(ORF)has 1 026 base pairs,encoding 341 amino acids.In comparison of its nucleotide and amino acid sequences with those of other baculovirus,HearMNPV v-cath shows the highest homology with that of Mamestra configurata NPV-B(MacoNPV-B),but the lowest homology with that of Cydia pomonella GV(CpGV).It is therefore suggested that HearMNPV v-cath had two ways of evolution:one is point mutation and the other is short nucleotide sequences.

Elucidating the evolution of Baculoviridae is essential to understanding the overwhelming diversity of virus.In the article some information about the evolution of Baculoviridae were described at detail,these items including the analysis method of single gene phylogeny and complete genome analyses,the relationships between evolution and variation,the baculovirus-integrated transposon,coevolutionary interactions with their hosts.

素质教育对教师的教育观念和学生的学习方法都提出了挑战。本文积极探索将协作式学习中校企合作的特点和研究性教学中创新教育的理念有机结合,贯穿于课堂教学环节、课外调研活动、企业实践环节以及科研模拟训练等多个层面,尝试改观高校传统教学的弊端,满足培养学生创新能力的迫切需要。

The 30K protein was successfully expressed in the Bombyx mori L.(Bm)cells by baculovirus system.B.mori cells and larvae were infected with the recombinant baculovirus(Bm/r-30K),respectively.The viability of cells and larvae were assayed at various times post-infection and BmPAK6 was used as a control.The viability of BmN cells infected with baculovirus(Bm/r-30K)was higher than that of the cells infected with BmPAK6 virus,the viability and surviving time of B.mori larvae were increased as well.The results indicate that over-expression of the B.mori 30K gene contributes to increase the surviving time of BmN cells and larvae.

为研究30K蛋白抑制细胞凋亡的作用,利用RT-PCR从家蚕总RNA中扩增到30KC19基因,对其进行了序列分析并克隆到杆状病毒转座载体pFastBacHTb中,构建成重组转座载体pFastBacHTb-30KC19。利用杆状病毒（Bac-to-Bac）表达系统筛选重组杆状病毒,在昆虫细胞TN5中进行了表达和蛋白检测。结果表明30K蛋白在昆虫细胞中得到了高效表达。

通过对酿造酱油中 G+、 G-菌组成情况的分析,研究了天然生物防腐剂 Nisin对酿造酱油中腐败细菌的抑制效力.对腐败细菌在不同 Nisin含量的营养肉汤培养基及酱油原液中培养试验的结果显示:在腐败菌最适生长温度 36℃下培养,当 Nisin的含量达到 100μ g/ mL时,营养肉汤 (pH4.60)中腐败细菌的总数急剧减少;当 Nisin的含量达到 50～ 100μ g/mL时,即能有效地抑杀酱油中的腐败细菌.这表明 Nisin可替代化学防腐剂用于酱油的防腐保鲜,以减轻酱油的巴氏灭菌强度,保持酱油原有营养成分、风味、色泽,延长制品货架期.

通过对酿造酱油中G+、G-菌组成情况的分析,研究了Nisin对酿造酱油中腐败细菌的抑制效力.对腐败细菌在不同Nisin含量的营养肉汤培养基及酱油原液中培养试验的结果显示:在腐败菌最适生长温度36℃下培养,当Nisin的含量达到100μg/ml时,营养肉汤(pH4.60)中腐败细菌的总数急剧减少;当Nisin的含量达到50～100μg/ml时,即能有效地抑杀酱油中的腐败细菌.这表明Nisin可替代化学防腐剂用于酱油的防腐保鲜,以减轻酱油的巴氏灭菌强度,保持酱油原有营养成分、风味、色泽,延长制品货架期.