Microsatellite Genotyping for Four Expected Inbred Mouse Strains from KM Mice

Chinese Kun Ming （KM） mouse, an outbreed strain of laboratory animal, has been widely utilized in related pharmaceutical and genetic studies throughout China. However, the value of KM mice to the research community has been severely limited, partially due to the fact that well-characterized inbred strain of KM mice is not available. Several expected inbred strains from KM mice have been bred, but their genetic purity remains uncertain. In this study, four expected inbred strains of KM mice （A1, T2, N2, and N4） were chosen and their inbred degree were compared with two classical inbred mouse lines （BALB/c and C57BL/6） by analyzing the genotypes of about 30 microsatellite markers. In the four strains, A1 and N4 were homozygous at all genotyped loci, but N2 and T2 were only heterozygous at locus D15Mit16. These results indicate that the level of genetic purity/homozygousity of A1, N4, N2, and T2 inbred line is comparable to those of BALB/c and C57BL/6. This study provided the first and solid evidence for genetic purity of four expected inbred strains of KM mice. These 4 inbred mice strains should be well maintained for further characterization and utilization in genetic studies.

Characteristics of gut microbiota in representative mice strains:Implications for biological research

Background:Experimental animals are used to study physiological phenomena,pathological mechanisms,and disease prevention.The gut microbiome is known as a potential confounding factor for inconsistent data from preclinical studies.Although many gut microbiome studies have been conducted in recent decades,few have focused on gut microbiota fluctuation among representative mouse strains.Methods:A range of frequently used mouse strains were selected from 34 isolation packages representing disease-related animal(DRA),immunity defect animal(IDA),or gene-editing animal(GEA)from the BALB/c and C57BL/6J backgrounds together with normal mice,and their microbial genomic DNA were isolated from mouse feces to sequence for the exploration of gut microbiota.Results:Mouse background strain,classification,introduced source,introduced year,and reproduction type significantly affected the gut microbiota structure(p<0.001 for all parameters),with background strain contributing the greatest influence(R^(2)=0.237).In normal groups,distinct gut microbiota types existed in different mouse strains.Sixty-four core operational taxonomic units were obtained from normal mice,and 12 belonged to Lactobacillus.Interestingly,the gut microbiota in C57BL/6J was more stable than that in BALB/c mice.Furthermore,the gut microbiota in the IDA,GEA,and DRA groups significantly differed from that in normal groups(p<0.001 for all).Compared with the normal group,there was a significantly higher Chao 1 and Shannon index(p<0.001 for all)in the IDA,GEA,and DRA groups.Markedly changed classes occurred with Firmicutes and Bacteroidetes.The abundances of Helicobacter,Blautia,Enterobacter,Bacillus,Clostridioides,Paenibacillus,and Clostridiales all significantly decreased in the IDA,GEA,and DRA groups,whereas those of Saccharimonas,Rikenella,and Odoribacter all significantly increased.

Prepulse inhibition (PPI) of tactile startle response in recombinant congenic strains of mice:QTL mapping and comparison with acoustic PPI

Prepulse inhibition （PPI） of the startle response is a psychophysiological measure of sensorimotor gating believed to be cross-modal between different sensory systems. We analyzed the tactile startle response （TSR） and PPI of TSR （tPPI）, using light as a prepulse stimulus, in the mouse strains A/J and C57BL/6J and 36 recombinant congenic strains derived from them. Parental strains were significantly different for TSR, but were comparable for tPPI. Among the congenic strains, variation for TSR was significant in both genetic backgrounds, but that of tPPI was significant only for the C57BL/6J background. Provisional mapping for loci modulating TSR and tPPI was carried out. Using mapping data from our previous study on acoustic startle responses （ASR） and PPI of ASR （aPPI）, no common markers for aPPI and tPPI were identified. However, some markers were significantly associated with both ASR and TSR, at least in one genetic background. These results indicate cross-modal genetic regulation for the startle response but not for PPI, in these mouse strains.

Establishment of a Transgenic Mice Model Specifically Expressing 172￣(His) Mutant p￣(53)in Mammary Gland

The codon 172His of murine p53 was mutated from CGC to CAC by using recombinant PCR, and the amino-acid coded was correspondingly changed from arginine to histidine. The 172His mutant p53 was cloned in the cassette of rat WAP promoter and SV40 poly (A) signal to make WAP-172￣(His) mutant p53-SV40 transgene construct. This transgene was microinjected into the fertilized eggs of FVB and C57BL mice. Of the totally 32 Fo mice, 9 were identified by unique PCR as 172His mutant p53 transgenic mice. Mutant p53 transgene was overexpressed in that mammary gland of three transgenic mice and expressed at lower level in the mammary gland of another two transgenic mice. The overexpression of 172His mutant p53 resulted in the incidcnce of mammary tumor in mousc #8512 of 11 months old. So far, the 172His mutant p53 transgene and its expression in the mammary gland have been already transmitted up to F8.

Protective effects of cyclosporine A on T-cell dependent ConA-induced liver injury in Kunming mice

INTRODUCTIONThe T-cell dependent specific liver injury in mice induced by concanavalin A(ConA) is a newly cstablished experimental liver injury model,which is considered more eligible for the study on pathophysiology of several human liver discascs,such as viral hepatitis and autommune hepatitis[1-9].T cell activation and several cytokines release had been proven to play a critical role in ConA -induced liver injury[10-19].Cyclosprine A(CsA),an effective inhibitor of activation of T lymphocytc,hes been used widely in clinical treatment,especially in autoimmune diseases and organ transplantation[20-25].In this study,we investigated the possible effect of CsA on ConA-induced liver injury in Kunning mice.

Molecular mechanism about lymphogenous metastasis of hepatocarcinoma cells in mice

AIM: To investigate the correlation between lymphogenous metastasis and matrix metalloproteinases (MMPs) activity and the expression of Fas ligand of tumor cells in lymph nodes. METHODS: Fifty-six inbred 615-mice were equally divided into 2 groups and inoculated with Hca-F and Hca-P cells. Their lymph node metastatic rates were examined. Growth fraction of lymphocytes in host lymph nodes was detected by flow cytometry. The Hca-F and Hca-P cells were cultured with extract of lymph node, liver or spleen. The quantity of MMPs in these supernatants was examined by zymographic analysis. The expression of Fas ligand, PCNA, Bcl-2 protein of Hca-F and Hca-P cells in the mice were examined by immunohistochemistry. The apoptosis signals of macro-phages in lymph nodes were observed with in situ DNA fragmentation. RESULTS: On the 28th day post-inoculation, the lymph node metastatic rate of HcaF was 80%(16/20), whereas that of Hca-P was 25%(5/20). The growth fraction of lymphocytes was as follows: in the Hca-F cells, the proliferating peak of lymphocytes appeared on the 14th day post inoculation and then decreased rapidly, while in HcaP cells, the peak appeared on the 7th day post inoculation and then kept at a high level. With the extract of lymph node, the quantity of the MMP-9 activity increased (P【0.01) and active MMP-9 and MMP-2 were produced by both Hca-F and Hca-P tumor cells, which did not produce MMPs without the extract of lymph node or with the extracts of the liver and spleen. The expression of Fas Ligand of Hca-F cells was stronger than that of Hca-P cells (P 【0.01). The expressions of PCNA and Bcl-2 protein of Hca-F cells in the tumors of inoculated area were the same as that of Hca-P cells. In situ DNA fragmentation showed that the positive signals of macrophages were around Hca-F cells. CONCLUSION: Secretion of MMPs which was associated with metastatic ability of Hca-F and Hca-P tumor cells depends on the environment of lymph nodes. The increased expression of Fas ligand protein of Hca-F tumor cells with high lymphogenous metastatic potential in lymph nodes may help tumor cells escape from being killed by host lymphocytes.

Effects of Exposure to Electromagnetic Field on of Some Hematological Parameters in Mice

Objective: To investigate the effects of electromagnetic fields on some hematological parameters of Male white mice. Methods: In this study two experiments were used as short-term and long-term experiments. Short-term experiments were carried out in 80 male white Balb/c mice exposed to two types of mobile phone for a period time up to 60 min a day for 2 weeks. Long-term experiments was carried out in 30 male white Balb/c mice exposed to two types of mobile phone for a period time up to 90 min a day for a day for a month, two months, three months. Result: The present result found that a decline in hemoglobin, hematocrit, red blood cells count, in addition to the platelets count after short and long exposure to both types of mobile phone (Alcatel, Nokia,). It was observed that the average number of white cells and lymphocytes increased significantly, indicating the increase to the body’s immune response to radiation. Also, when exposed to both devices (Alcatel, Nokia) for 15 minutes, the red blood cells began to take roles formation due to an increase in the viscosity of the blood and the ability of the cells adhesion. Increased duration of exposure to 30-minute to both devices revealed disparities in the sizes of red blood cells with the appearance of a large proportion of cells with pale colors due to lack of hemoglobin. In the group exposed for 45 minutes showed clearly the influence of mobile phones on examining blood smears which observed a proportion variation in the cell sizes with the emergence of forms of abnormal forms, including pellets cleft with many areas empty of red blood cells, With increasing in of exposure duration to 60 minutes, red blood cells appeared completely different from the natural form and took the forms resembling the eye and tear that appear in the case of anemia. After three months of exposure to both types of mobile phone showed pathological changes in red blood cells where gripped the outer membrane of the red corpuscle changes and become serrated. Conclusion: Exposure to electromagnetic fields is responsible for the variations of some hematological parameters in rats.

Screening of Saccharomyces Strains Highly Producing Glutathione and Breeding of Its Ethionine-resistant Mutants

[Objective] The aim of this study was to screen Saccharomyces for glutathione over-production. [Method] Ethionine-resistant mutants were obtained through UV mutagenesis and rational screening. [Result] A high GSH-producing strain HSJB1 was isolated from soil, and the biomass for this strain by flask shaking fermentation was 3.87 g/L while the GSH yield was 91.87 mg/L. According to the morphological, physiological and biochemical characteristics of cells, this strain was primarily identified as Saccharomyces cerevisiae. An ethionine-resistant mutant YBS77 was obtained through UV mutagenesis of the original strain HSJB1, and the biomass for this strain by flask shaking fermentation was 7.60 g dry cell weight/L while the GSH yield was 211.96 mg/L. [Conclusion] The biomass of the mutant obtained by breeding is increased by 96.38% than that of the original strain, and the GSH yield of the mutant obtained by breeding is increased by 130.72% than that from the original strain, which indicates that the breeding method is feasible.

Analysis of genetic relationship in mutant silkworm strains of Bom-byx mori using inter simple sequence repeat (ISSR) markers

Amplified inter simple sequence repeats （ISSR） markers were used to determine genetic relationships among mutant silkworm strains of Bombyx mori. Fifteen ISSR primers containing simple sequence repeat （SSR） motifs were used in this study. A total of 113 markers were produced among 20 mutant strains, of which 73.45% were found to be polymorphic. In selected mutant genetic stocks, the average number of observed allele was （1.7080 ± 0.4567）, effective alleles （1.5194 ± 0.3950） and genetic diversity （Ht） （0.2901 ± 0.0415）. The dendrogram produced using the unweighted pair group method with arithmetic means （UPGMA） and cluster analysis made using Nei＇s genetic distance resulted in the formation of one major group containing 6 groups separated 20 mutant silkworm strains. Therefore, ISSR amplification is a valuable method for determining the genetic variability among mutant silkworm strains. This efficient molecular marker would be useful for characterizing a considerable number of silkworm strains maintained at the germplasm center.

美人鱼发光杆菌美人鱼亚种dly基因缺失株构建及其生物学特性研究

美人鱼发光杆菌美人鱼亚种(Photobacterium damselae subsp.damselae,PDD)是广泛分布于全球海洋环境中的一种致病菌。本研究以实验室保存的一株高致病性PDD菌株(PDD1608)作为对象,初步探究毒力基因dly对PDD菌株的生物学特性和致病力的影响。利用λRed重组技术成功构建毒力基因dly缺失株Δdly PDD1608::Cm,比较野生株和缺失株的生长、涌动性、药物敏感性、生理生化特性、生物被膜形成能力、菌株及胞外产物(extracellular products,ECP)的溶血性和磷脂酶活性等生物学特性。选用海水青鳉鱼(Oryzias melastigma)作为实验动物,通过人工感染实验测定野生株和缺失株及其ECP对海水青鳉鱼的致病性。结果显示,毒力基因dly缺失后导致PDD菌株生长变慢,涌动性、溶血性和磷脂酶活性均降低;野生株和缺失株的药物敏感性和生理生化特性未产生变化;与野生株相比,缺失株的生物被膜形成能力有显著差异(P<0.05);人工感染实验表明,缺失株及其ECP对海水青鳉鱼的致病性降低。毒力基因dly影响PDD菌株的生长、涌动性、溶血性和磷脂酶活性等多种生物学特性,并且与PDD菌株及其ECP的致病力强弱密切相关。

HSV-1突变株M6感染人支气管上皮细胞后对巨噬细胞介导的免疫反应的影响

目的探究1型单纯疱疹病毒(herpes simplex virus type 1,HSV-1)突变株M6感染人支气管上皮细胞(16HBE细胞)后对巨噬细胞介导的免疫反应的影响。方法用HSV-1感染16HBE细胞分析培养液中细胞因子的变化;将巨噬细胞与被HSV-1毒株感染的16HBE细胞的上清液共培养并通过尾静脉回输至小鼠体内,分别在第1、3、7、28、56、90天对小鼠淋巴结细胞因子表达水平、脾脏T细胞比例变化、小鼠中和抗体表达水平以及特异性T细胞反应进行检测。结果16HBE细胞被HSV-1突变株感染后,上清液中募集和激活巨噬细胞相关的细胞因子均较高水平表达但略低于野毒株组;尾静脉回输实验后,突变株组小鼠淋巴结炎症因子、趋化因子和T细胞的比例随时间发生了不同的变化,并引起了弱于野毒株组的体液免疫和强于野毒株组的特异性T细胞免疫反应,且仅极少数与野毒株组具有显著性差异(P<0.05)。结论16HBE细胞被HSV-1突变株M6感染后能够释放募集和激活巨噬细胞的细胞因子,使巨噬细胞携带HSV-1突变株的特异性活化信息,激活了宿主的免疫系统,诱导了宿主的体液免疫和细胞免疫。

成骨细胞条件性视黄酸信号失活小鼠模型的构建与验证

目的·构建并验证可模拟维生素A缺乏症(vitamin A deficiency,VAD)导致的颅颌面骨畸形且出生后可存活的模型小鼠。方法·运用Cre-LoxP重组酶系统,通过将Osx^(Cre)与Rosa26dnRARα/dnRARα基因型的小鼠多代杂交,构建成骨细胞视黄酸受体α显性负突变体(dominant-negative retinoid acid receptorαmutation,dnRARα)条件性过表达小鼠Osx^(Cre);Rosa26^(dn/dn),实现成骨细胞视黄酸信号条件性失活以模拟VAD状态。取Osx^(Cre);Rosa26^(dn/dn)小鼠及其同窝对照小鼠股骨骨髓间充质干细胞(bone mesenchymal stem cell,BMSC)与颅顶骨细胞,成骨诱导后,采用蛋白质印迹法(Western blotting)验证成骨细胞中视黄酸受体α(retinoid acid receptorα,RARα)蛋白的表达水平。取Osx^(Cre);Rosa26^(dn/dn)小鼠及其同窝对照小鼠颅顶骨细胞经诱导形成的成骨细胞,采用双荧光素酶报告基因技术验证视黄酸信号通路抑制程度。同时分别应用表达Cre、eGFP的腺病毒(Ad-eGFP与Ad-Cre)感染Rosa26^(dn/dn)小鼠股骨BMSC与颅顶骨细胞,成骨诱导后运用相同方法评价显性负突变蛋白表达水平与视黄酸信号通路抑制程度。取6周龄小鼠颅骨,运用Micro-CT及三维重建技术验证Osx^(Cre);Rosa26^(dn/dn)模型小鼠颅颌面骨畸形表型。结果·Western blotting结果显示,Osx^(Cre);Rosa26^(dn/dn)小鼠股骨及颅顶骨成骨细胞相较同窝对照小鼠表现出RARα蛋白水平上调。双荧光素酶报告基因实验结果显示Osx^(Cre);Rosa26^(dn/dn)小鼠成骨细胞的视黄酸反应元件(retinoid acid response element,RARE)活性相较同窝对照小鼠下调(P<0.05)。同时,Ad-Cre感染后Rosa26^(dn/dn)小鼠股骨及颅顶骨成骨细胞相较Ad-eGFP感染后表现出RARα蛋白水平上调,成骨细胞的RARE活性下调(P<0.05)。Micro-CT及三维重建结果显示,6周龄Osx^(Cre);Rosa26^(dn/dn)小鼠颅骨呈现长度缩短、骨发育不全等VAD样颅颌面骨畸形。结论·成功构建了成骨细胞条件性视黄酸信号失活小鼠模拟VAD所致颅颌面骨畸形,为未来VAD所致颅颌面骨畸形出生后致病机制与潜在靶点的研究提供了新的模型与途径。

利用末龄幼虫蜕和蛹壳对苹果蠹蛾基因的无创伤检测方法

苹果蠹蛾Cydia pomonella(L.)是我国重大农业入侵害虫,对我国果业健康发展造成严重威胁。在利用基因编辑等技术对相关基因进行功能研究时,通常采用单对交配策略对纯合突变体进行筛选。因此,在配对前明确个体基因型,避免对虫体造成损伤尤为重要。本研究分别收集末龄幼虫蜕(10个蜕)和蛹壳(1个、5个、10个、15个蛹壳),通过基因组DNA提取、目的基因PCR扩增、琼脂糖凝胶电泳检测和PCR产物测序,以评估该方法作为苹果蠹蛾基因检测的可行性。结果显示,以苹果蠹蛾末龄幼虫10个蜕及5个以上蛹壳提取的基因组DNA作为模板,扩增精巢特异性丝氨酸/苏氨酸蛋白激酶(Testis specific serine/threonine protein kinase,TSSK1)基因,PCR产物经琼脂糖凝胶电泳检测得到单一、明亮的条带,经测序证实该产物是目的基因序列。基于高效及节约成本的原则,拟推荐利用10个末龄幼虫蜕或10个蛹壳提取基因组DNA、通过PCR扩增和测序进行苹果蠹蛾的无损伤基因型检测。本研究建立了一种利用幼虫蜕和蛹壳进行苹果蠹蛾无损伤、高效的基因检测方法,为提高苹果蠹蛾基因功能研究的效率奠定了基础。

超声应变技术评估PCSK9基因敲除对肥胖小鼠心肌功能的影响

目的应用超声应变技术评估PCSK9基因敲除对肥胖小鼠心肌功能的影响,并探讨其机制。材料与方法选取6周龄野生型C57BL/6雄性小鼠20只,随机分为正常组和肥胖组,各10只;选取6周龄PCSK9^(-/-)C57BL/6雄性小鼠10只为PCSK9^(-/-)组。肥胖组和PCSK9^(-/-)组给予高脂饲料造模,正常组给予普通饲料,12周后从肥胖组和PCSK9^(-/-)组中选取造模成功小鼠,3组小鼠行心脏超声、透射电镜及免疫荧光染色进行观察。结果肥胖组、正常组和PCSK9^(-/-)组小鼠室间隔舒张末期厚度分别为(0.98±0.13)mm、(0.77±0.07)mm、(0.78±0.13)mm,差异有统计学意义(F=5.10,P=0.02),肥胖组较正常组增厚(t=2.73,P<0.05),PCSK9^(-/-)组较肥胖组变薄(t=-2.92,P<0.05)。3组左心室整体纵向应变及整体圆周应变差异有统计学意义(F=7.44、15.40,P<0.05),其中肥胖组较正常组整体纵向应变和整体圆周应变减低(t=3.79、5.50,P<0.05),PCSK9^(-/-)组较肥胖组整体纵向应变和整体圆周应变升高(t=-2.53、-3.37,P<0.05)。电镜及免疫荧光结果显示,肥胖组心肌超微结构受损,NLRP3及Caspase-1表达较正常组增多(t=12.53、-4.73,P<0.05),PCSK9^(-/-)组心肌超微结构受损明显改善,NLRP3及Caspase-1表达较肥胖组减少(t=-6.23、2.05,P<0.05)。结论超声应变技术较常规心脏超声参数能更敏感地发现PCSK9基因敲除肥胖小鼠心肌功能的改变;PCSK9基因敲除后可能通过抑制NLRP3及Caspase-1表达改善肥胖小鼠心肌功能。

Biological Background of Kh.DIC Mice and Their Learning and Memory Defects

The learning ability of the Kh.DIC mice, a mutant of the Kunming mice, was studied to analyze its memory development. The mice's brain function was evaluated using a water maze with the amount of monoamines measured by fluorospectrophotometry and enzyme activities detected by ultraviolet spectrophotometry. The mice were found to have spacial learning and memory defects at the age of 1 month in both ordinary animals and in special pathogen free (SPF) animals. At the same time, the amount of monoamines and the activities of monoamine oxidase B and dopamine β hydroxylase differed from those of the Kunming mice. The defects might be related to the differences in the monoamine neurotransmitter system. The results suggest that the DIC mice may be useful economic animal models for the study of brain defects.

新型冠状病毒抗体和小分子抑制剂的研究现状

世界卫生组织已宣布新型冠状病毒感染(coronavirus disease 2019,COVID-19)的爆发为全球大流行。中和抗体和小分子抑制剂在预防及治疗COVID-19中发挥重要作用。尽管已开发出了多种中和抗体以及疫苗,但是随着病原体严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)的不断变异,现有的抗体及疫苗面临巨大的挑战。小分子抑制剂主要通过干扰病毒与宿主的结合以及病毒自身的复制达到消灭病毒以及抑制病毒感染的作用,并且对SARS-CoV-2突变株具有广谱抑制作用,是当前研究的热点。近年来国内外学者对SARS-CoV-2的小分子抑制剂做了大量的研究工作,本文根据中和抗体识别的抗原表位以及小分子抑制剂的作用机制分别对用于预防及治疗COVID-19的中和抗体和小分子抑制剂进行综述,讨论其研究现状,并展望小分子抑制剂的应用前景,以期为该领域的进一步研究提供参考。

PEDV G2突变株动力学特征比较研究

[目的]本研究旨在通过对猪流行性腹泻病毒(PEDV)经连续传代后的毒株(PEDV-BJ-150)进行全面研究,探究其生物学特性、遗传变异、致病能力和细胞适应性,以评估其作为潜在减毒疫苗株的潜力。[方法]通过对PEDV-BJ-150和原始PEDV-BJ-01毒株进行细胞培养、动物感染试验和基因组测序,评估其在不同细胞系中的增殖能力、病毒滴度、致病性以及基因组的核苷酸序列变异并利用动物模型评估其感染特性和病理变化。[结果]PEDV-BJ-150经连续传代后在Vero-M细胞中表现出更高的增殖能力和滴度,尤其在悬浮适应株Vero-M和贴壁适应株Vero-M中表现出显著的滴度差异。实验结果显示其在体内感染后的致病性降低,尤其在肺部未检测到病毒存在,鼻翼和小肠部位显示更高的病毒滴度。此外,基因组测序结果表明PEDV-BJ-150的S蛋白存在多个位置的氨基酸突变,可能影响其细胞嗜性和致病能力。[结论]本研究发现经连续传代的PEDV-BJ-150在体内表现出的毒力和致病能力明显降低,表明其有作为减毒疫苗株的潜力。特别是S蛋白中的连续性氨基酸缺失可能导致病毒致病性降低,这为疫苗研发提供了新的思路。此外,研究发现不同组织对PEDV的感染特性不同,这对于深入了解病毒传播机制具有重要意义。本研究的创新之处在于发现了PEDV经连续传代后的致病性变化和S蛋白突变对病毒特性的影响,为病毒进化和疫苗设计提供了新的见解。这项研究为了解PEDV的病毒学特性和毒力变化提供了重要线索,对于病毒疫苗研发和应对疫情具有潜在的应用前景。

Biodegradation of Phenol and 4-Chlorophenol by the Mutant Strain CTM 2

The biodegradations of phenol and 4-chlorophenol （4-cp） were studied using the mutant strain CTM 2 obtained by the He-Ne laser irradiation on wild-type Candida tropicalis. The results showed that the capacity of the CTM 2 to biodegrade 4-cp was increased up to 400 mg·L^-1 within 59.5 h. In the dual-substrate biodegradation both velocity and capacity of the CTM 2 to degrade 4-cp increased with low-concentration phenol. A totalof 400 mg·L^-1 4-cp was completely degraded within 50.＇5 h in thepresence of 300 mg·L^-1 phenol. The maximum 4：cp biodegegradation could reach 440 mg·L^1 with 120 mg·L^1 phenol. Low-concentration 4-cp caused great inhibition on the CTM 2 to degrade phenol. In addition, the kinetic behaviors were described using the kinetic model proposed in this lab.

Construction and expression of eukaryotic plasmids containing lamivudine-resistant or wild-type strains of Hepatitis B Virus genotype C

AIM:To construct eukaryotic expression plasmids of full-length Hepatitis B Virus(HBV) genotype C genome,which contain lamivudine-resistant mutants(YIDD,YVDD) or wild-type strain(YMDD) ,and to observe the expression of HBV DNA and antigens [hepatitis B surface antigen(HBsAg) and hepatitis B e antigen(HBeAg) ] of the recombinant plasmids in HepG2 cells. METHODS:Three HBV full-length genomes were amplified from the plasmids pMD18T-HBV/YIDD,pMD18T-HBV/YVDD and pMD18T-HBV/YMDD,using PCR. Three recombinant plasmids were generated by inserting each of the PCR products into the eukaryotic expression vector pcDNA3.1(+) ,between the EcoRI and HindⅢ sites. After being characterized by restriction endonuclease digestion,and DNA sequence analysis,the recombinant plasmids were transfected into HepG2 cells. At 48 and 72 h post-transfection,the levels of intracellular viral DNA replication were detected by real-time PCR,and the expression of HBsAg and HBeAg in the cell culture supernatant was determined by ELISA. RESULTS:Restriction endonuclease digestion and DNA sequence analysis confirmed that the threerecombinant plasmids were correctly constructed. After transfecting the plasmids into HepG2 cells,high levels of intracellular viral DNA replication were observed,and HBsAg and HBeAg were secreted into the cell culture supernatant. CONCLUSION:Eukaryotic expression plasmids pcDNA3.1(+) -HBV/YIDD,pcDNA3.1(+) -HBV/YVDD or pcDNA3.1(+) -HBV/YMDD,which contained HBV genotype C full-length genome,were successfully constructed. After transfection into HepG2 cells,the recombinant plasmids efficiently expressed HBV DNA,HBsAg and HBeAg. Our results provide an experimental basis for the further study of HBV lamivudine-resistant mutants.

Production and Accumulation of Xylooligosaccharides with Long Chains by Growing Culture and Xylanase of a Mutant Strain of Bacillus pumilus X-6-19

Bacillus pumilus X-6-9 isolated from soil and subsequently identified, produced xylooligosaccharides with long chains from xylan and accumulated them in the culture. By improving the culture conditions and mutating the bacterium, a 3.2-fold increase in the production of the xylooligosaccharides was established, when compared to the original culture conditions of B. pumilus X-6-19. The addition of D-glucose to the culture of the mutant strain U-3 of B. pumilus X-6-9 repressed the synthesis of β-xylosidase, but not xylanase. Thus, it was revealed that strain U-3 was a good organism for the production and accumulation of xylooligosaccharides with long chains from xylan by a microbial culture. Xylanase produced by strain U-3 was purified to homogeneity and characterized. The hydrolyzates generated by the purified xylanase contained xylobiose, xylotriose, xylotetraose, and xylopentaose, but not xylose.