肺炎支原体P1蛋白羧基端基因片段在大肠杆菌中的表达及应用研究

目的:克隆、表达和鉴定肺炎支原体(Mycoplasma pneumoniae,MP)P1蛋白羧基端基因序列,为制备抗体和基因工程疫苗打下基础。方法 在成功克隆肺炎支原体P1蛋白羧基端基因片段并测序的基础上,将基因序列克隆到表达载体pET32a(+)上,构建了重组表达质粒pET32a(+)/P1(3 520~4 563bp),转化大肠杆rosetta,IPTG诱导表达,利用Ni^2＋亲和层析柱对重组蛋白进行纯化,并用ELISA和Western blotting方法检测其抗原性。重组蛋白免疫小鼠,制备单克隆抗体。结果 重组蛋白在大肠杆菌中可以高效表达,SDS-PAGE显示其相对分子质量与预计大小一致,蛋白质纯度达95％以上。ELISA和Western blotting实验证实,重组蛋白具有良好的抗原性。并成功获得两株高效价单克隆抗体。结论:本研究成功克隆和表达了肺炎支原体P1蛋白羧基端基因序列,制备了抗肺炎支原体P1蛋白单克隆抗体,为肺炎支原体诊断试剂和疫苗的开发等进一步的研究奠定了基础。     

Development of a polymerase chain reaction assay for Mycoplasma salivarium by using the nucleotide sequence within aminopeptidase My gene

A polymerase chain reaction assay for a 278-nucleotide DNA fragment within aminopeptidase My gene of Mycoplasma salivarium was developed. The assay amplified M. salivarium DNA, but did not amplify DNAs of other mollicutes, bacteria and mammalian cells. The detection limit of the assay was 10 fg of DNA, approximately equivalent to 10 organisms.     

The adhesin related 30-kDa protein of Mycoplasma pneumoniae exhibits size and antigen variability

Mycoplasma pneumoniae is a pathogenic bacterium colonizing epithelial cells of the human respiratory tract. Using an erythrocyte binding assay we isolated a cytadsorption negative mutant designated M7 which has lost 12 of a total of 13 repetitive sequences of a proline rich C-terminal region of the adhesin related 30-kDa protein. The truncated adhesin related protein of 22 kDa showed reduced antigenicity compared to the corresponding wild-type protein. Moreover, the mutant M7 proved incapable of adhering to erythrocytes and to a human colon carcinoma cell line indicating that the repetitive C-terminal region of the 30-kDa protein is essential for effective cytadherence. The adhesin related 30-kDa protein as well as the truncated forms of the corresponding protein were accessible to carboxypeptidase Y which clearly shows surface exposure of the C-terminus of this protein.     

猪肺炎支原体Mhp367蛋白不同区段与恢复期血清反应能力的比较

【背景】课题组前期研究发现猪肺炎支原体Mhp367蛋白是体液免疫显性蛋白,但该蛋白不同区段与猪肺炎支原体恢复期血清的反应能力尚不明确。【目的】鉴定Mhp367蛋白不同区段与猪肺炎支原体恢复期血清的反应能力。【方法】利用不同的引物组合扩增mhp367基因片段,扩增的片段连接pGEX-6P-1、pGEX-4T-3或pGEX-5X-3载体,转化大肠杆菌DH5α感受态细胞。提取的质粒经Bam H I和Xho I双酶切及测序确定重组质粒是否构建成功。正确的重组质粒转化大肠杆菌BL21(DE3)感受态细胞构建重组菌。重组菌经IPTG诱导和超声破菌后,经与谷胱甘肽beads结合和SDS-PAGE电泳检测目的蛋白表达情况。表达目的蛋白的重组菌破菌后上清包被谷胱甘肽板,ELISA方法鉴定Mhp367蛋白不同区段与猪肺炎支原体恢复期血清的反应能力。【结果】构建了9个能以可溶形式表达目的蛋白的重组菌;9个Mhp367蛋白片段均为体液免疫显性,第394-524位氨基酸区段与猪肺炎支原体恢复期血清反应最强,是一个良好的疫苗候选抗原区段。【结论】本研究为猪肺炎支原体基因工程亚单位疫苗的研发提供了候选抗原靶标。     

滑液囊支原体不同感染途径的致病力比较

【背景】自2010年以来,我国鸡群中滑液囊支原体(Mycoplasma synoviae,MS)的感染率不断增高,目前MS已广泛存在于我国不同的鸡群中,包括蛋鸡、种鸡、白羽肉鸡和地方品种鸡等,其血清阳性率已超过40%,严重危害我国养鸡业,并造成了严重的经济损失。【目的】系统比较MS以不同感染途径对56日龄无特定病原体(Specific Pathogen Free,SPF)鸡的致病力。【方法】采用MS强毒FZ株以点眼、爪垫注射、胸部皮下注射、单次气管注射、连续3次气管注射等不同途径感染56日龄SPF鸡,观察感染后临床症状和解剖病理变化,检测感染后血清中的MS抗体,并且对气管组织进行病原再分离和组织病理学观察。【结果】MS的FZ株以不同途径感染SPF鸡后临床表现和发病率差异较大,爪垫注射和胸部皮下注射可导致100%的鸡发生爪垫肿大或胸部囊肿,而单次或连续3次气管注射可引起33%-50%的鸡发生严重气囊炎,点眼感染途径基本不能引起临床病理变化;爪垫肿大主要为肉芽组织增生和出现大量的黄色干酪样块状物质,胸部囊肿在囊肿部位有大量的血红色样液体和黄色干酪样块状物质;组织病理学结果显示连续3次气管注射方式感染更易对气管造成损伤,表现为气管黏膜固有层/黏膜下层发生轻微至轻度灶性炎细胞浸润,而爪垫肿大和胸部囊肿组织有大量的纤维组织和血管增生,同时伴有大量炎性细胞浸润;点眼和气管注射途径的气管MS病原再分离率可达100%,而胸部皮下注射或脚垫注射也可从气管中分离到MS病原;爪垫注射途径更易引起MS抗体转阳。【结论】系统比较了MS以不同感染途径对56日龄SPF鸡的致病性,并筛选出了相应的评价指征,成功建立了MS人工感染8周龄SPF鸡的发病模型,其中点眼感染途径和气管注射途径以气管病原再分离作为主要指征,辅以气囊炎进行评价,而胸部皮下注射和爪垫注射途径分别以胸部囊肿和爪垫肿胀作为主要病理指征进行评价。     

猪肺炎支原体与宿主相互作用研究进展

猪肺炎支原体(Mycoplasma hyopneumoniae,Mhp)是猪支原体肺炎(Porcine enzootic pneumonia,PEP)的病原体。由于难以建立基因操作平台,也没有成熟的动物模型,这为Mhp致病机制研究增加了极大的困难。但支原体学家仍然在Mhp与宿主互作方面取得了一定进展。文中从Mhp对宿主细胞的黏附、损伤、刺激宿主产生的炎症反应和免疫反应4个方面进行了综述,并对今后Mhp致病机理研究方向进行了展望,以期为后续的Mhp与宿主互作研究提供借鉴,为有效疫苗和药物开发提供理论依据。     

Metal-induced extracellular protein excretion in Arthrobacter aurescens

Abstract Mycoplasma contamination of cell cultures is a menace to diagnostic and research procedures. Rapid and reliable detection methods are, therefore, sorely needed. After comparing 16S rRNA sequences from those mycoplasmas that contaminate cell cultures, three different oligonucleotide probes were constructed. Two of these probes were designed to be group-specific and one to be species-specific. The three oligonucleotide probes were designed to cover all mycoplasmas commonly isolated from cell cultures. Contaminated cell lines could easily be detected by a direct filter hybridization assay in which the probes were incubated jointly. The assay proved to be rapid and sensitive with the possibility to perform and evaluate the mycoplasma testing within one working day.     

Analysis of deep sequencing exosome-microRNA expression profile derived from CP-II reveals potential role of gga-miRNA-451 in inflammation

Mycoplasma gallisepticum (MG) can cause chronic respiratory disease (CRD) in chickens. While several studies have reported the inflammatory functions of microRNAs during MG infection, the mechanism by which exosomal miRNAs regulate MG-induced inflammation remains to be elucidated. The expression of exosome-microRNA derived from MG-infected chicken type II pneumocytes (CP-II) was screened, and the target genes and function of differentially expressed miRNAs (DEGs) were predicted. To verify the role of exosomal gga-miR-451, Western blot, ELISA and RT-qPCR were used in this study. The results showed that a total of 722 miRNAs were identified from the two exosomal small RNA (sRNA) libraries, and 30 miRNAs (9 up-regulated and 21 down-regulated) were significantly differentially expressed. The target miRNAs were significantly enriched in the treatment group, such as cell cycle, Toll-like receptor signalling pathway and MAPK signalling pathway. The results have also confirmed that gga-miR-451-absent exosomes derived from MG-infected CP-II cells increased inflammatory cytokine production in chicken fibroblast cells (DF-1), and wild-type CP-II cell–derived exosomes displayed protective effects. Collectively, our work suggests that exosomes from MG-infected CP-II cells alter the dynamics of the DF-1 cells, and may contribute to pathology of the MG infection via exosomal gga-miR-451 targeting YWHAZ involving in inflammation.     

Complex interactions between bacteria and haemosporidia in coinfected hosts: An experiment

1. Hosts are typically coinfected by multiple parasite species whose interactions might be synergetic or antagonistic, producing unpredictable physiological and pathological impacts on the host. This study shows the interaction between Plasmodium spp. and Leucocytozoon spp. in birds experimentally infected or not infected with Mycoplasma gallisepticum.
2. In 1994, the bacterium Mycoplasma gallisepticum jumped from poultry to wild birds in which it caused a major epidemic in North America. Birds infected with M. gallisepticum show conjunctivitis as well as increased levels of corticosterone.
3. Malaria and other haemosporidia are widespread in birds, and chronic infections become apparent with the detectable presence of the parasite in peripheral blood in response to elevated levels of natural or experimental corticosterone levels.
4. Knowing the immunosuppressive effect of corticosterone on the avian immune system, we tested the hypothesis that chronic infections of Plasmodium spp. and Leucocytozoon spp. in house finches would respond to experimental inoculation with M. gallisepticum as corticosterone levels are known to increase following inoculation.
5. Plasmodium spp. infection intensity increased within days of M. gallisepticum inoculation as shown both by the appearance of infected erythrocytes and by the increase in the number and the intensity of positive PCR tests.
6. Leucocytozoon spp. infection intensity increased when Plasmodium spp. infection intensity increased, but not in response to M. gallisepticum inoculation. Leucocytozoon spp. and Plasmodium spp. seemed to compete in the host as shown by a negative correlation between the changes in their PCR score when both pathogens were present in the same individual.
7. Host responses to coinfection with multiple pathogens measured by the hematocrit and white blood cell count depended on the haemosporidian community composition. Host investment in the leukocyte response was higher in the single‐haemosporidia‐infected groups when birds were infected with M. gallisepticum.
8. A trade‐off was observed between the immune control of the chronic infection (Plasmodium spp./Leucocytozoon spp.) and the immune response to the novel bacterial infection (M. gallisepticum).

     

Mollicute Anti-Adhesive and Growth Inhibition Properties of the Methanolic Extract of Propolis from the Brazilian Native Bee Melipona quadrifasciata

Hydroalcoholic propolis extracts from the bee species Melipona quadrifasciata have been shown to possess antimicrobial activity against different mollicute strains, but a methanolic extract (ME) could contain an increased diversity of nonpolar bioactive components with a potentially higher antimicrobial activity. The ME obtained by maceration of the propolis sample was fractionated with solvents of different polarities and then, purified by silica gel column chromatography through biomonitoring of its antimicrobial activity against mollicute strains. Analysis by gas chromatography-mass spectrometry (GC/MS) enabled the identification of compounds using the NIST library. Minimum inhibitory concentrations (MICs) of the samples were determined by broth microdilution. Anti-adhesive assays were performed with Mycoplasma pneumoniae cells. The hexane (MIC=62.5 mg/L) and dichloromethane (MIC=125 mg/L) fractions presented the most promising results against M. pneumoniae. They were fractionated into 74 subfractions, and even the best ones did not show better results (MIC>250 mg/L) than their original fractions, likely due to the loss of terpene compounds that seem to act in synergy. The dichloromethane subfraction FD4 was highlighted in the anti-adhesive assay with an inhibitory activity of 21.6 %. A synergistic effect of the nonpolar compounds in M. quadrifasciata propolis may be responsible for its antibacterial activity, but several purified components can improve its anti-adhesive properties.