尼罗罗非鱼无乳链球菌Sip蛋白乳酸菌活载体口服疫苗的研制及其免疫效果

链球菌病是威胁我国罗非鱼养殖产业健康发展的重要病害之一。为研制出免疫效果好、操作简便的罗非鱼链球菌病疫苗,本研究构建重组表达无乳链球菌Sip蛋白的穿梭质粒pNZ8124-Sip,通过酶切和测序验证后电转化乳酸乳球菌NZ9000,获得能够诱导重组表达无乳链球菌Sip蛋白的乳酸菌活菌载体疫苗。采用SPS-PAGE电泳摸索最佳诱导浓度和诱导时间以获得最大表达量,通过镍柱纯化目的蛋白并进行Western blot检测;利用不同浓度的重组乳酸菌活载体疫苗灌胃口服免疫尼罗罗非鱼,采用间接ELISA法测定免疫后血清抗体水平变化,通过人工腹腔注射感染无乳链球菌获得相对免疫保护率。研究结果显示,构建的重组乳酸乳球菌可通过nisin诱导表达大小为48 ku特异性蛋白,与目的蛋白大小一致;PAGE电泳显示,重组蛋白主要以可溶蛋白和包涵体2种形式存在,其中胞内可溶性蛋白浓度达7.65 mg/mL;诱导表达的最佳条件为100 ng/mL nisin诱导6 h;Western blot检测结果显示,诱导蛋白可与鼠抗His标签抗体特异性结合。口服免疫结果显示,中浓度组(2.24×10~(10) CFU/mL)和低浓度组(2.24×10~9 CFU/mL)免疫2次能够显著提高尼罗罗非鱼的血清抗体水平和抗无乳链球菌感染能力,中浓度免疫组的相对免疫保护率最高为41.0%。本研究可为罗非鱼链球菌病口服疫苗的研究奠定基础,具有广阔的应用前景。     

Effect of a potential probiotics Lactococcus garvieae B301 on the growth performance,immune parameters and caecum microflora of broiler chickens

In this study, a novel Lactococcus garvieae B301 was isolated from the intestinal tract of a healthy piglet. L. garvieae B301 was tolerant to acid pH, simulated gastric and small intestinal transit juices, indicating that it was capable of surviving in the gastrointestinal tract. L. garvieae B301 was safe and beneficial to broilers, as broiler chickens supplemented with L. garvieae B301 had lower diarrhoea incidence and mortality than the Control. Moreover, supplementation of broiler diets with L. garvieae B301 resulted in an increase in body weight and the number of caecum lactic acid bacteria and Bifidobacterium spp., and decrease in feed‐to‐gain ratio and the number of caecum coliforms. It also had a positive effect on the thymus index and bursa of Fabricius index and enhanced serum levels of immune globulins. All these results showed that L. garvieae B301 could enhance the growth performance of broiler chickens and improve their health. Thus, L. garvieae B301 could be a promising feed additive for broiler chickens.     

乳酸乳球菌高效电转化方法的建立

以乳酸菌诱导型表达质粒pNZ8149为载体,Lactococcus lactisNZ3900为受体,采用电转化方法研究电场强度、电击杯规格、受体菌株的生长状态、感受态细胞浓度及分装体积、质粒浓度、电击脉冲时间以及电击杯使用次数等因素对电转化效率的影响。结果表明：在固定电阻200Q、电容25μF条件下,收获对数生长前期的乳球菌制作感受态细胞,并以80μL体积分装,采用2mm规格的电击杯,加入浓度为1．2μg／μL的质粒,在电场强度和脉）中时间分别为10kV／cm和5ms的条件下完成电击转化。转化后的菌液恢复培养2h后涂板计数,转化效率最高可以达到2．6×10^4CFU／μgDNA。研究结果为进一步构建乳酸菌高效表达系统和食品级基因工程乳酸菌株奠定了方法基础。     

Lancefield group C Streptococcus dysgalactiae infection responsible for fish mortalities in Japan

A Lancefield serological group C Streptococcus sp. was isolated from cultured amberjack, Seriola dumerili Risso, and yellowtail, Seriola quinqueradiata Temminck and Schlegel, immunized with Lactococcus garvieae commercial vaccines in Japan. The isolated bacteria were Gram-positive cocci, auto-aggregating in saline, morphologically long chains in growth medium, catalase negative and alpha-haemolytic on blood agar. An almost complete gene sequence of the 16S rDNA of two isolates was determined and compared with that of bacterial strains in the database. The isolates were identified as Streptococcus dysgalactiae based on the results of the 16S rDNA sequence, the bacteriological properties and the Lancefield serological grouping. Oligonucleotide primers specifically designed for the 16S-23S rDNA intergenic spacer region of S. dysgalactiae amplified a gene from all the fish isolates, as well as the type strains alpha-haemolytic S. dysgalactiae subsp. dysgalactiae ATCC430738 and beta-haemolytic S. dysgalactiae subsp. equisimilis ATCC35666, but not those of S. equi ATCC33398, Lactococcus garvieae ATCC43921 and L. garvieae KG9408. The severe necrotic lesions of the caudal peduncle seen in experimentally infected fish were similar to those seen in naturally infected fish.     

诱变育种提高乳酸乳球菌Lactococcus lactis产Nisin的能力

为了获得高产细菌素的乳酸乳球菌菌株,通过采用紫外线(UV)、亚硝基胍(NTG)、硫酸二乙酯(DES)诱变和紫外线与硫酸二乙酯复合诱变的方法,获得多株诱变菌株,研究突变株代谢生产Nisin的效价及抑菌能力。结果表明,其中通过诱变得到了一株高产菌株DU101,该菌株代谢生产Nisin的效价由442 IU/m L提高到了1386 IU/m L,约是原始菌株HDBR-06的3.14倍。通过传代试验验证了菌株的遗传性能稳定。     

Lactococcus garvieae Endocarditis on a Prosthetic Biological Aortic Valve

Lactococcus garvieae (LG) endocarditis is a rare disease in humans. There are only about 16 reported cases in the world. We report a 76‐year‐old male patient with LG endocarditis. In depth interview with the patient revealed that 2 weeks prior to admission, he had eaten sushi containing raw fish. Unlike many of the other infections reported, which were on a native mitral valve, our patient's vegetation was on a prosthetic aortic valve.     

Development of a novel PCR assay based on the 16S-23S rRNA internal transcribed spacer region for the detection of Lactococcus garvieae

Lactococcus garvieae is recognized as an emerging pathogen in fish. Several PCR‐based methods have been developed for the detection and identification of L. garvieae; however, the sensitivity of these methods is still in question regarding the discrimination of this organism from other closely related species. Two primers, ITSLg30F and ITSLg319R, were designed from the sequence in the 16S–23S internal transcribed spacer (ITS) region and used for the specific detection of L. garvieae. L. garvieae strains including fish isolates were positive by this method. In contrast, previously developed PCR methods showed false‐positive results with non‐L. garvieae species. Our results indicate that a PCR method using the newly designed ITS primer set provides a sensitive and efficient tool for the detection of L. garvieae in fish and aquaculture environments.     

乳酸乳球菌抗氧化的研究进展

乳酸菌(Lactic acid bacteria,LAB)主要是通过发酵产能的,但是有研究发现,在添加氯化高铁血红素后乳酸乳球菌(Lactococcus lactis)能形成一条呼吸链,在乳酸乳球菌(L.lactis)IL1403中发现了能够编码呼吸链蛋白的基因,同样的现象在粪链球菌中(Strepto coccus feacalis)也有发现。当氧出现在电子传递链中,质子的排出和质子运动势(PMF)的产生都会加倍。相比较其他的兼性或者专性厌氧菌,乳酸乳球菌(L.lactis)的呼吸作用可以促进代谢能量的产生,由于这种呼吸作用,乳酸乳球菌(L.lactis)有更好的生长得率和显著的存活率,这表明在发酵条件下产生的代谢能量不足,从而会限制乳酸乳球菌(L.lactis)的生长。氧在呼吸链中扮演了重要的有益角色,而不是起毒害作用。     

Construction of a Food-Grade Expression Vector Based on pMG36e by Using an α-Galactosidase Gene as a Selectable Marker

Construction of a food-grade expression vector for application to lactic acid bacteria（LAB） is of importance for dairy fermentation system. An α-galactosidase（aga） gene encoding an enzyme degrading melibiose was amplified by PCR from the plasmid p RAF800 of Lactococcus lactis NZ9000. The aga gene was introduced into pMG36 e to substitute the p rimary antibiotic selectable marker of pMG36 e, resulting in construction of a new food-grade expression vector pMG36-aga. To testify the expression efficiency of exogenous gene in pMG36-aga, a 1.5 kb long α-amylase（amy） gene from Ba cillus li cheniformis was cloned by PCR and introduced into the plasmid pMG36-aga. The resultant plasimd pMG36-aga-amy was transformed into L. lactis ML23 by electroporation. The positive clones were selected with the medium containing melibiose as the sole carbon source. Th e selection efficiency of aga was 8.71×103 CFU with a standard deviation of 9.1×102 CFU ？g-1 DNA of pMG36-aga. Furthermore, the SDS-PAGE analysis showed that the pMG36-aga-amy expressed a 56.4 kDa protein which was the same as the putati ve molecular weight of α-amylase. The starch plate assay also indicated that L. lactis ML23 displayed high activity of α-amylase by expressing of amy gene of pMG36-aga-amy.