CRISPR/Cas9系统在寄生虫基因组编辑中的应用研究进展

基因编辑是一种依赖于核酸内切酶对目标基因进行定向改造的现代生物学技术,可实现特定片段的加入、删除,以及特定碱基的插入、缺失、替换等。CRISPR/Cas9系统(clustered regularly interspaced short palindromic repeats/associated nuclease 9)是在细菌和古细菌中发现的一种用来抵御外源质粒和噬菌体入侵的获得性免疫系统,经改造后已成为一种新型的基因组编辑工具,因其具有操作简单、成本低、切割效率高等优点被广泛应用于多种动物、植物及微生物等的基因组编辑。应用CRISPR/Cas9系统在进行基因编辑的基础上可分析和验证基因功能、研究遗传育种及筛选药物靶点和疫苗分子等,以达到减弱病原微生物致病性、增强动植物抗病性、治疗遗传性疾病、病毒性感染及肿瘤等目的。寄生虫因其生活史复杂,与细菌和病毒相比基因组较大、结构较复杂,缺乏有效的研究工具等原因,使得相关研究进展缓慢,而CRISPR/Cas9基因编辑技术的出现为基因功能研究提供了新的技术手段。笔者对CRISPR/Cas9系统的发现及其在寄生虫基因组编辑中的应用研究进展进行综述,并...     

Molecular Characterization of a H9N2 Subtype Swine Influenza Virus

The objective of this study was to explore the epidemic situation and pathogenic characteristics of swine influenza virus (SIV) in Shandong Province. In the spring of 2019, 130 swine nasal swab samples were collected from a slaughterhouse in Tai'an city, Shandong Province for virus isolation and identification. The whole genome of isolated virus was sequenced and analyzed. Meanwhile, 1 527 swine serum samples were collected from swine farms in 8 regions of Shandong province and their anti-SIV antibody were detected by HI assay using standard avian H9N2 antigen. The results showed that a H9N2 subtype influenza virus strain was isolated and named as A/swine/Shandong/TA009/2019(H9N2). The homology analysis showed that the isolated virus had close genetic relationship with A/environment-air/Kunshan/NIOSH-BL20/2018(H9N2) and A/environment-air/Kunshan/NIOSH-BL25/2018(H9N2), and the nucleotide homology of the gene fragments were above 99.5%. Phylogenetic analysis results demonstrated that HA and NA genes of the isolated virus belong to the Y280-like lineage, PB2 and M genes belong to the G1-like lineage, and PB1, PA, NP and NS genes belong to the SH/F98-like lineage. The cleavage site in HA protein is “PSRSSR/GL”, which was in accordance with the molecular biological characteristics of low pathogenic avian influenza virus.The position 216 of HA protein is L, and it has the ability to bind human-derived sialic acid α 2,6-Gal. The results of HI showed that 9 among 1 527 serum samples were positive with a positive rate of 0.59%. The isolated virus was swine-derived H9N2 virus, and serological investigations revealed that H9N2 subtype virus infection was present in swine herds in Shandong Province. The results of this study suggest that continuous surveillance of the SIV epidemiological situation and its pathogenic characteristics should be strengthened.     

Two cases of acute polyradiculoneuritis in dogs consuming a raw poultry diet

A 9-year-old female mixed-breed dog presented with ascending flaccid tetraparesis, and a 5-year-old castrated male Poodle dog presented with ventroflexion of neck, dysphonia, and hindlimb weakness, which progressed to acute ascending tetraparesis. Both dogs were fed raw poultry for over 9 and 5 years, respectively. Blood examination and other test results were normal or unrelated to the present case. Fecal polymerase chain reaction analysis in the Poodle dog was positive for Clostridium perfringens and Campylobacter jejuni. Tetraparesis improved with supportive care in both dogs. Human IV immunoglobulin was only administered to the Poodle dog, which showed a shorter recovery (12 days compared to 34 days in the mixed-breed dog). Both dogs returned to normal conditions eventually.     

利用PCR技术同时检测番茄抗根结线虫基因(Mi-1)和抗叶霉病基因(Cf-9)

   利用同一PCR反应体系,对分别与番茄抗根结线虫的Mi-1基因和抗番茄叶霉病的Cf-9基因紧密连锁的CAPS(cleaved amplified polymorphic sequences)标记进行同时扩增筛选,扩增的特异性片段与单引物扩增片段吻合与Cf-9基因紧密连锁的CAPSl标记在抗病试材中可扩增出2775 bp的特异片段,且存在Taq I酶切位点,酶切后产生1177 bp、446 bp、370 bp和160 bp的不同特异片段;与Mi-1基因紧密连锁的CAPS2为共显性标记,抗性材料中产生915 bp的特异片段,Taq I酶切后产生719bp和196 bp的特异片段该体系可用于在同一PCR反应体系中对Mi-1和Cf-9 2个抗病基因进行同时筛选鉴定该体系的建立不仅省时、省工,节省费用,而且可用于苗期早期辅助选育,加快番茄育种进程     

Reactions of sera from laboratory, domestic and wild animals in Africa with Protein a and a recombinant chimeric Protein AG

An ELISA was developed to determine the reactivity of peroxidase labelled Protein A and a recombinant Protein A + Protein G construct, to sera from a variety of laboratory, domestic and wild animals from Africa. There was variability in the binding capacity of sera from individuals of the same species, but four groups could be recognized. Sera from birds and crocodiles were at most weakly reactive with either Protein A or the chimeric construct. Sera from some domestic animals such as horse, goat and cat, and sera from some wild ungulates including buffalo, wildebeest, waterbuck and impala were reactive with Protein A, but reacted to a much greater degree with the chimeric construct. Sera from larger wild animals such as elephant, rhinoceros and giraffe were strongly reactive with the chimeric protein and moderately reactive with Protein A. Sera from primates and dog, pig, guinea pig and rabbit reacted strongly with both proteins. Chimeric proteins that combine the IgG binding capacities of Protein A and Protein G can be used to detect immunoglobulin from a wide variety of African wild animal species. They may thus be of great value in seroepidemiological investigations of these animal populations.     

牦牛血低聚肽对小鼠抗疲劳能力和缺氧H9c2细胞的影响

为研究牦牛血低聚肽对缺氧所致H9c2心肌细胞损伤以及对运动后小鼠抗疲劳能力的影响,本研究通过建立H9c2细胞缺氧损伤模型以及小鼠负重游泳试验,研究其抗缺氧能力以及抗疲劳能力。结果表明,与正常对照组相比,缺氧组的H9c2细胞存活率极显著降低(P<0.01),表明缺氧对H9c2心肌细胞有一定损伤;与缺氧组相比,低聚肽干预组的细胞存活率增加且存在剂量依赖性;与常用抗缺氧药物红景天苷相比,牦牛血低聚肽高剂量组细胞存活率为75.1%,而红景天苷组为88.0%。牦牛血低聚肽及其复合肽均能延长小鼠负重游泳时间,提高运动后小鼠肝脏肝糖元(HG)含量,降低运动后小鼠血清乳酸(BLA)和血清尿素氮(BUN)含量。综上,牦牛血低聚肽对缺氧介导的心肌细胞损伤具有一定的保护作用,能提高小鼠的抗疲劳能力,且牦牛血低聚肽与大豆低聚肽按照5∶5比例复配后的抗疲劳能力优于单一使用牦牛血低聚肽。本研究结果为耐缺氧活性因子的研究提供了一条新途径。     

含山梨醇的新型生物有机肥促生效应与机理研究

向含解淀粉芽孢杆菌SQR9的生物有机肥中添加山梨醇研制了新型生物有机肥,并通过黄瓜盆栽和拟南芥培养试验研究了该新型生物有机肥的促生效应与机理。结果表明,含解淀粉芽孢杆菌SQR9的生物有机肥对黄瓜具有显著促生效果,添加山梨醇有利于促进解淀粉芽孢杆菌SQR9在土壤中定殖,能进一步提高生物有机肥的促生效应。施用该新型生物有机肥能显著提高土壤中速效养分的含量以及黄瓜对养分的吸收。山梨醇能有效促进菌株SQR9分泌生长素（吲哚乙酸,IAA）,进而促进野生型拟南芥根系的生长,而在使用IAA不敏感突变体时促生效果消失,说明山梨醇促进有益菌SQR9分泌IAA是该新型生物有机肥显著促进作物生长的机理之一。本研究提出了新型生物有机肥研发的新思路,为植物促生菌新应用模式的开发提供了理论依据。     

表没食子儿茶素没食子酸酯和维生素C联用对高尿酸血症小鼠血尿酸水平的影响

以KM雄性小鼠为研究对象,酵母膏（7.5 g·kg^-1）和氧嗪酸钾（250 mg·kg^-1）联合给药建立高尿酸血症小鼠模型,探究表没食子儿茶素没食子酸酯（EGCG）和维生素C（Vc）联用对高尿酸血症小鼠血尿酸水平的影响。将小鼠随机分为6组（n=6）：空白组、模型组、别嘌呤醇组（阳性药组）、EGCG组、EGCG联合Vc组和Vc组,连续给药7 d后测定生化指标。结果表明,与模型组相比,EGCG联合Vc组小鼠的血尿酸值（UA）,血尿素氮（BUN）和肌酐（Cr）水平均明显降低（P<0.001）;EGCG与Vc联用明显抑制了肝脏中腺苷脱氨酶（ADA）和黄嘌呤氧化酶（XOD）的活性（P<0.05或P<0.01）,并显著下调了肾脏中葡萄糖转运蛋白9（GLUT9）mRNA的表达（P<0.001）;肾脏切片显示EGCG和Vc联用显著改善高尿酸血症小鼠的肾脏损伤。此外,EGCG与Vc联用对高尿酸血症小鼠的作用效果优于EGCG。     

116个小麦品种（系）抗叶锈基因Lr9-Lr26、Lr19-Lr20的复合PCR检测

[目的]建立简单、快速、有效的小麦抗叶锈基因复合PCR体系,从而提高分子标记辅助选择效率。[方法]以28个‘Thatcher’为背景的近等基因系和16个已知基因载体品系作为试材,测试了小麦抗叶锈病基因Lr9、Lr26、Lr19和Lr20的STS标记特异性,通过优化PCR反应体系和循环条件,构建了抗叶锈基因Lr9-Lr26和Lr19-Lr20的复合PCR检测体系。对116个小麦品种(系)所含有的抗叶锈病基因进行了分子检测。[结果]供试品种均不含有Lr9和Lr20,47个品种含有Lr26(基因频率为40.5%),‘中梁22’含有Lr19。经反复验证,Lr9-Lr26和Lr19-Lr20复合PCR技术检测结果可靠,且与上述单个分子标记检测结果一致。[结论]建立的Lr9-Lr26和Lr19-Lr20的复合PCR检测体系可以准确、稳定、高效地检测小麦抗叶锈基因Lr9、Lr26、Lr19和Lr20。     

Interactions between viral infection and immune mechanisms

Viruses differ from other infectious agents mainly in two respects: (a) they may be generated in the genome of normal cells and, in other instances, reinserted into the genome as proviruses, (b) they may participate in all pathologic processes that form the main subject of immune surveillance and immunity such as infectious diseases, tumourous growth, and, auto-aggressive processes.

Due to intracellular and cell modifying character of virus infection, the immune mechanisms are often unsuccessful in elimination of the agent from the vertebrate organism and may show disturbances contributing to development of a persistent viral disease. As exemplified by two forms of leprosy, intracellular bacteria may also cause ‘slow’ diseases. Their course, in analogy with viral slow diseases, seems to reflect the changed immunological potential of the infected host.