pcDNA3.1-HPV18E7重组基因的克隆

目的 从HPV18感染细胞中提取HPV18E7 DNA,构建pcDNA3．1-HPV18E7重组质粒。方法 应用PCR方法从HPV18感染细胞中扩增HPV18E7 DNA,并应用基因重组技术构建pcDNA3．1-HPV18E7重组质粒。结果 PCR扩增产物经电泳检测及测序鉴定证实为HPV18E7;基因重组产物经PCR鉴定及测序证实为pcDNA3．1-HPV18E7,成功构建了pcDNA3．1-HPV18E7重组质粒。结论 从受感染人体细胞中成功扩增出HPV18E7,并成功构建pcDNA3．1-HPV18E7重组质粒,为HPV基因疫苗的构建和治疗HPV感染相关肿瘤奠定了基础。     

重组人表皮生长因子应用于肛裂手术的临床研究

目的：研究外源性表皮生长因子对于肛裂手术后的手术创面愈合情况的影响。方法：肛裂手术后，治疗组患者每日用重组人表皮生长因子敷于肛管创面，对照组患者采用常规的方式换药。比较两组患者术后伤口疼痛、出血情况，以及伤口肉芽生长愈合情况。结果：治疗组在减轻术后伤口疼痛、伤口水肿和出血等方面均显著优于对照组，其平均住院时间和伤口愈合时间也显著短于对照组。结论：外源性表皮生长因子有助于肛裂术后创面的快速修复，减轻患者的痛苦。     

中国东北部延边地区丙型肝炎病毒1a型感染研究

目的初步探讨东北部延边地区HCV基因型分布和1a型的感染状况。方法对44例来自延边地区的HCVRNA阳性样本进行HCV5’非编码区（5’NCR）复合酶切分型分析。将分型结果为1a型的4例样品（分别为Y2、Y4、Y6、Y8）进行5’NCR和NS5B区的扩增,测序,然后与27个HCV全基因参考序列（均来自GenBank）比对并构建遗传进化树。结果44例样品中1a／1b混合型19例（43．1％）,1b型12例（27．3％）,2a／1b混合型8例（18．2％）,1a型4例（9．1％）,2a型1例（2．3％）,2b型以及3～6型未检出。其中,1a型的4例样品Y2、Y4、Y6、Y8分别与各基因型的全基因参考序列相比较,在5’NCR与1a型参考序列HC—J1的同源性最高,分别为0．990、0．990、0．990、0．990,进化树分析也证实为1a型;在NS5B区与1b型参考序列HC—J4的同源性最高,分别为0．936、0．957、0．936、0．936,进化树分析表明为1b型。结论延边地区以1a／1b型混合感染为主,1b型和1b／2a型感染次之,与中国其他地区存在明显差异。对4例1a型HCV病毒株的分析发现,存在HCV5’NCR与NS5B分型结果不一致现象。分析推测可能是HCV基因组在生物进化过程中自然重组的表现,但尚需进一步研究证实。     

Recent advances in lipid nanoparticles for delivery of nucleic acid,mRNA, and gene editing-based therapeutics

Lipid nanoparticles (LNPs) are becoming popular as a means of delivering therapeutics, including those based on nucleic acids and mRNA. The mRNA-based coronavirus disease 2019 vaccines are perfect examples to highlight the role played by drug delivery systems in advancing human health. The fundamentals of LNPs for the delivery of nucleic acid- and mRNA-based therapeutics, are well established. Thus, future research on LNPs will focus on addressing the following: expanding the scope of drug delivery to different constituents of the human body, expanding the number of diseases that can be targeted, and studying the change in the pharmacokinetics of LNPs under physiological and pathological conditions. This review article provides an overview of recent advances aimed at expanding the application of LNPs, focusing on the pharmacokinetics and advantages of LNPs. In addition, analytical techniques, library construction and screening, rational design, active targeting, and applicability to gene editing therapy have also been discussed.     

细粒棘球蚴延伸因子-1重组蛋白对小鼠的免疫保护性研究

目的:探讨细粒棘球蚴(Eg)延伸因子(Eg.EF-1)重组蛋白对小鼠的免疫保护性及其作为候选疫苗的潜在价值。方法:ICR小鼠随机分为免疫组和佐剂对照组,每隔2周皮下免疫1次,在第3次免疫后2周,用Eg原头节进行攻击感染,感染后20周剖杀小鼠,检获Eg包囊,计算免疫保护力,并用酶联免疫吸附法测定血清中免疫球蛋白(Ig)G及其亚型和IgE水平。结果:与佐剂对照组比较,免疫组小鼠的免疫保护力为85.6%,血清IgG、IgG1、IgG2a水平显著性升高(P<0.05),IgG2b水平轻微降低(P>0.05),IgG3和IgE水平无明显变化。结论:rEg.EF-1重组蛋白能诱导小鼠产生一定的免疫保护力,是潜在的疫苗候选抗原分子。     

芽孢杆菌外毒素保护性抗原与上游强启动子穿梭载体的构建

目的 构建携带炭疽芽孢杆菌保护性抗原（PA）的两种穿梭载体与上游强启动子。方法 将解淀粉芽孢杆菌的α-淀粉酶启动子克隆到载体pblueseript—sk（＋）上，构建载体pBLKSP；以A16R疫苗株（Tox＋，Cap-，弱毒株）DNA为模板，设计合成内外侧引物巢式PCR扩增获得保护性抗原（PA）的全基因，先克隆到载体pBLKSP，再将其和质粒PUB110重组构建成两种穿梭载体。结果 酶切鉴定显示所切下的片段大小均与预计相符。测序结果与文献报道序列及预计结果一致。结论 成功构建了带有强启动子的两种穿梭载体。为在无毒炭疽疫苗株中的高效表达和炭疽芽孢杆菌的分子疫苗研究奠定了基础。     

表达人JNK基因重组腺病毒的构建和鉴定

目的 制备表达人c-jun氨基末端激酶(JNK)复制缺陷型重组腺病毒.方法 将重组穿梭载体pAdTrack-CMV-WT-JNK线性化后,与pAdEasy-1共转化大肠杆菌BJ5138,进行同源重组得到重组腺病毒载体.将重组腺病毒载体转染入包装细胞HEK293内制备复制缺陷型重组腺病毒,并经PCR及DNA测序鉴定.结果 JNK重组腺病毒载体能有效转染HEK293细胞并在细胞内成功包装,5 d后可以观察到绿色荧光蛋白(GFP)明显表达,搜集的病毒经过PCR扩增得到特定JNK基因片段并测序鉴定.动物实验证实构建的Ad-WT-JNK能有效在肝组织表达.结论 该研究成功构建了JNK重组腺病毒载体及相应重组腺病毒颗粒,为进一步研究JNK的作用及应用JNK进行相关疾病的基因治疗奠定了基础.     

Formation and stability of linear plasmids in a recombination deficient strain of yeast

Summary Natural termini from macronuclear DNA of the ciliated protozoans Tetrahymena thermophila and Oxytricha fallax can support telomere formation in yeast. However, plasmids carrying these ciliate termini are modified by the addition of DNA which hybridizes to the synthetic oligonucleotide poly [d(C-A)], a sequence which also hybridizes to terminal restriction fragments from yeast chromosomes but not to Tetrahymena or Oxytricha macronuclear DNAs. Thus, in yeast, the creation of new telomeres on ciliate termini involves the acquisition of yeast-specific terminal sequences presumably by either recombination or non-templated DNA synthesis. The RAD52 gene is required for the majority of yeast mitotic and meiotic recombination events. Moreover, the absence of an active RAD52 gene product results in high rates of chromosome loss. Here we demonstrate that terminal restriction fragments from Tetrahymena macronuclear ribosomal DNA (rDNA) support the formation of modified telomeres in a yeast strain carrying a defect in the RAD52 gene. Moreover, linear plasmids bearing these modified ciliate termini are stably propagated in rad52 ^– cells.     

非何杰金淋巴瘤免疫表型和基因型分析

用单克隆抗体免疫酶法和体外基因扩增聚合酶链反应(PCR)技术,研究了25例非何杰金淋巴瘤(NHL)免疫表型及其中8例免疫球蛋白和T细胞受体(TCR)基因重排的基因型。结果表明,表达B系细胞分化抗原者14例中,5例起源于前B细胞,9例起源于晚期成熟B细胞;表达T系细胞分化抗原者9例中,起源于幼稚或成熟胸腺细胞者各4例、普通胸腺细胞者1例。另有2例同时表达了T,B系细胞分化抗原。8例基因分析者中,4例表达B系和3例表达T系细胞分化抗原者分别检测到IgH和TCRγ基因克隆性重排,各有1例发生TCRγ和IgH基因系间交叉重排。1例表达T,B系细胞分化抗原者出现IgH基因重排和TCRδ基因缺失。讨论了非何杰金淋巴瘤免疫表型和基因重排分析的临床应用价值。     

Common RNA replication signals exist among group 2 coronaviruses: evidence for in vivo recombination between animal and human coronavirus molecules

5' and 3' UTR sequences on the coronavirus genome are known to carry cis-acting elements for DI RNA replication and presumably also virus genome replication. 5' UTR-adjacent coding sequences are also thought to harbor cis-acting elements. Here we have determined the 5' UTR and adjacent 289-nt sequences, and 3' UTR sequences, for six group 2 coronaviruses and have compared them to each other and to three previously reported group 2 members. Extensive regions of highly similar UTR sequences were found but small regions of divergence were also found indicating group 2 coronaviruses could be subdivided into those that are bovine coronavirus (BCoV)-like (BCoV, human respiratory coronavirus-OC43, human enteric coronavirus, porcine hemagglutinating encephalomyelitis virus, and equine coronavirus) and those that are murine hepatitis virus (MHV)-like (A59, 2, and JHM strains of MHV, puffinosis virus, and rat sialodacryoadenitis virus). The 3' UTRs of BCoV and MHV have been previously shown to be interchangeable. Here, a reporter-containing BCoV DI RNA was shown to be replicated by all five BCoV-like helper viruses and by MHV-H2 (a human cell-adapted MHV strain), a representative of the MHV-like subgroup, demonstrating group 2 common 5' and 3' replication signaling elements. BCoV DI RNA, furthermore, acquired the leader of HCoV-OC43 by leader switching, demonstrating for the first time in vivo recombination between animal and human coronavirus molecules. These results indicate that common replication signaling elements exist among group 2 coronaviruses despite a two-cluster pattern within the group and imply there could exist a high potential for recombination among group members.