大肠杆菌表达系统和酵母表达系统的研究进展

由于DNA重组技术和蛋白组技术的发展与成熟,外源基因表达技术备受关注。其在生物、食品、工业以及医学领域发挥着举足轻重的作用。以原核生物细胞及真核生物细胞作为表达系统,进行目的基因序列的表达以生产蛋白疫苗、核酸疫苗和生物酶制剂等是近年来发酵工业的新型领域。原核和真核表达系统是近年来研究和应用最广泛和最成熟的外源基因表达系统。对近年来关于大肠杆菌表达系统和酵母表达系统的研究进展进行了综述。     

秸秆纤维素分解细菌的筛选与鉴定

利用纤维素琼脂选择培养基和刚果红染色方法,从不同来源的土样中分离出2个菌株,测定了其纤维素酶活性、小麦秸秆降解能力,并对纤维素降解能力强的两个菌株进行了分子鉴定。结果表明,两株高效纤维素分解菌在纤维素刚果红培养基上均能形成透明水解圈,利用16S r RNA序列测序并分析Genbank中的同源性,结合形态和理化性质确定为Bacillus thuringiensis和Bacillus anthracis。在37℃,p H为7的条件下测定了纤维素酶活力,SC1-3和XR2-5-4分别为1.41和1.59。这两株37℃下经15 d后小麦秸秆失重率达19.92%和22.83%。     

Analysis of Inter-Individual Bacterial Variation in Gut of Cicada Meimuna mongolica (Hemiptera: Cicadidae)

Intestinal bacterial community plays a crucial role in the nutrition, development, survival, and reproduction of insects. When compared with other insects with piercing-sucking mouthparts, the habitats of cicada nymphs and adults are totally different. However, little is known about the differences in the gut bacterial communities in the nymphs and adults within any cicada species. The diversity of bacteria in the gut of nymphs and adults of both genders of Meimuna mongolica (Distant) was studied using the denaturing gradient gel electrophoresis (DGGE) method. Few inter-individual variations among gut microbiota were observed, suggesting that M. mongolica typically harbors a limited and consistent suite of bacterial species. Bacteria in the genera Pseudomonas and Enterobacter were the predominant components of the gut microflora of M. mongolica at all life stages. Bacteria of Pantoea, Streptococcus, and Uruburuella were also widespread in the cicada samples but at relatively lower concentrations. The relative stability and similarity of the PCR-DGGE patterns indicate that all individuals of this cicada species harbor a characteristic bacterial community which is independent from developmental stages and genders. Related endosymbionts that could be harbored in bacteromes of cicadas were not detected in any gut samples, which could be related to the cicada species and the distribution of these endosymbionts in the cicada cavity, or due to some of the possible limitations of PCR-DGGE community profiling. It is worthwhile to further address if related cicada endosymbiont clades distribute in the alimentary canals and other internal organs through diagnostic PCR using group-specific primer sets.     

Effect of hay type on cecal and fecal microbiome and fermentation parameters in horses

The effect of hay type on the microbiome of the equine gastrointestinal tract is relatively unexplored. Our objective was to characterize the cecal and fecal microbiome of mature horses consuming alfalfa or Smooth Bromegrass (brome) hay. Six cecally cannulated horses were used in a split-plot design run as a crossover in two periods. The whole plot treatment was ad libitum access to brome or alfalfa hay fed over two 21-d acclimation periods with subplots of sampling location (cecum and rectum) and sampling hour. Each acclimation period was followed by a 24-h collection period where cecal and fecal samples were collected every 3 h for analysis of pH and volatile fatty acids (VFA). Fecal and cecal samples were pooled and sent to a commercial lab (MR DNA, Shallowater, TX) for the amplification of the V4 region of the 16S rRNA gene and sequenced using Illumina HiSeq. The main effects of hay on VFA, pH, and taxonomic abundances were analyzed using the MIXED procedure of SAS 9.4 with fixed effects of hay, hour, location, period, and all possible interactions and random effect of horse. Alpha and beta diversities were analyzed using the R Dame package. Horses fed alfalfa had greater fecal than cecal pH (P ≤ 0.05), whereas horses fed brome had greater cecal than fecal pH (P ≤ 0.05). Regardless of hay type, total VFA concentrations were greater (P ≤ 0.05) in the cecum than in feces, and alfalfa resulted in greater (P ≤ 0.05) VFA concentrations than brome in both sampling locations. Alpha diversity was greater (P ≤ 0.05) in fecal compared with cecal samples. Microbial community structure within each sampling location and hay type differed from one another (P ≤ 0.05). Bacteroidetes were greater (P ≤ 0.05) in the cecum compared with the rectum, regardless of hay type. Firmicutes and Firmicutes:Bacteroidetes were greater (P ≤ 0.05) in the feces compared with cecal samples of alfalfa-fed horses. In all, fermentation parameters and bacterial abundances were impacted by hay type and sampling location in the hindgut.     

猪肠道细菌培养组学研究进展

猪肠道菌群是由所有定殖在猪肠道里的大量细菌、病毒、真菌和古菌等构成的集合。已有研究表明,很多疾病以及猪重要经济性状都与肠道菌群有关。目前,肠道菌群研究使用较多的技术是16S rRNA基因测序和宏基因组测序,但这些技术并不能了解具体菌株的实际功能和生理特性。肠道细菌的分离培养具有特殊重要的意义。近几年来,肠道细菌的培养取得了重要进展,基于培养条件的多样性分离培养出了更多的肠道细菌类别,这对促进肠道菌群在菌株水平的研究以及推广应用有重要意义。本文主要从猪肠道菌群组成结构、培养组学发展、猪肠道细菌培养组学的研究现状等方面进行论述和展望,为后续猪肠道菌群菌株的功能和影响表型的机制研究提供参考借鉴。     

玉米花丝多糖的提取制备与抑菌作用研究

本研究以玉米花丝为原料提取多糖。选取花丝类型、花丝和水的比例、温度和时间4个因素,采用L9(34)正交设计,两次重复。结果表明:玉米花丝多糖提取的最佳方法为:采用老花丝,花丝和水比例为1:6,60℃水浴2.5 h,提取率为3.65%。通过浓缩、离心、沉淀等过程,并去除蛋白质、色素等杂质,得到玉米花丝粗多糖。将粗多糖进行甲醇分级纯化,得到4种纯化多糖。再将4种多糖各配制成6种浓度,对活化后的8种细菌和4种真菌进行抑菌试验,测量抑菌圈直径,比较抑菌效果。结果表明:玉米花丝多糖对细菌有明显的抑制作用,对真菌也有一定的抑制作用,抑菌圈直径为11.5 mm。其中,以40%甲醇浓度,4000 r/min和45%甲醇浓度,5000 r/min进行离心分离的多糖对细菌和真菌的抑制效果较好。     

Cashew nut shell liquid potentially mitigates methane emission from the feces of Thai native ruminant livestock by modifying fecal microbiota

The methane-mitigating potency of cashew nutshell liquid (CNSL) was evaluated by investigating gas production from batch cultures using feces from Thai native ruminants that had been incubated for different periods. Feces was obtained from four Thai native cattle and four swamp buffaloes reared under practical feeding conditions at the Kasetsart University farm, Thailand. Fecal slurry from the same farm was also included in the analysis. CNSL addition successfully suppressed the methane production potential of feces from both ruminants by shifting short chain fatty acid profiles towards propionate production. Methane mitigation continued for almost 150 days, although the degree of mitigation was more apparent from Day 0 to Day 30. Bacterial and archaeal community shifts with CNSL addition were observed in feces from both ruminants; specifically, Bacteroides increased, whereas Lachnospiraceae and Ruminococcaceae decreased in feces to which CNSL was added. Fecal slurry did not show marked changes in gas production with CNSL addition. The findings showed that the addition of CNSL to the feces of ruminants native to the Southeast Asian region can suppress methane emission. Because CNSL can be easily obtained as a byproduct of the local cashew industry in this region, its on-site application might be ideal.     

Growth and morphologic response of rumen methanogenic archaea and bacteria to cashew nut shell liquid and its alkylphenol components

The growth and morphology of rumen methanogenic archaea (15 strains of 10 species in 5 genera, including 7 strains newly isolated in the present study) and bacteria (14 species in 12 genera) were investigated using unsupplemented in vitro pure cultures and cultures supplemented with cashew nut shell liquid (CNSL) and its phenolic compound components, anti-methanogenic agents for ruminant animals. Growth of most of the methanogens tested was inhibited by CNSL and alkylphenols at different concentrations ranging from 1.56 to 12.5 μg/ml. Of the alkylphenols tested, anacardic acid exhibited the most potent growth inhibition. Three gram-negative bacterial species involved in propionate production were resistant to CNSL and alkylphenols (>50 μg/ml). All the methanogens and bacteria that were sensitive to CNSL and alkylphenols exhibited altered morphology; disruption of the cell surface was notable, possibly due to surfactant activity of the tested materials. Cells division was inhibited in some organisms, with cell elongation and unclear septum formation observed. These results indicate that CNSL and alkylphenols, particularly anacardic acid, inhibit both rumen bacteria and methanogens in a selective manner, which could help mitigate rumen methane generation.     

Isolation and Identification of Pathogenic Bacteria Causing Dairy Cow Mastitis in Liaoning and Drug Sensibility Test of Pathogenic E.coli

In order to understand the main bacterial pathogen species causing dairy cow mastitis in Liaoning, as well as the characteristics of drug sensitivity of the pathogenic E.coli,the milk samples from 75 dairy cows with clinical manifestations for mastitis in certain large-scale dairy farm in Liaoning were collected.The bacteria in milk were cultured and isolated with biochemical methods and in vitro drug sensitivity tests were processed with the isolated E.coli strains.The results showed that the main bacterial pathogen for dairy cow mastitis were E.coli（separation rate 58.7%),S.aureus（64.0%）and S.agalactiae（54.7%),and multiple infection including double and triple infection were identified.The drug sensitivity tests on the isolated E.coli indicated that the E.coli isolates were highly resistant to sulfonamides（resistance rate>85%）and chloramphenicol（resistance rate>30%),and they were relatively low resistant to ampicillin（9.5%),ciprofloxacin（9.5%),ceftiofur（7.1%）and ofloxacin（4.8%).The results was able to provide reliable theoretical basis for prevention and control of dairy cow mastitis in Liaoning area.