人溶菌酶密码子优化及其在牛乳腺细胞中高效表达

【目的】分析牛乳腺中7种主要乳蛋白基因密码子使用偏好性,筛选高频密码子和低频密码子,依据其对人溶菌酶基因进行密码子局部优化和全局优化,通过牛乳腺上皮细胞等多种细胞对优化效果进行分析评价,为提高重组人溶菌酶表达量,开发新型、高效、安全的重组人溶菌酶提供理论依据。【方法】利用CodonW和EMBOSS等软件对7种主要牛乳蛋白和人溶菌酶基因密码子偏好进行生物信息学分析,筛选牛乳蛋白基因高频、低频密码子并根据牛乳蛋白基因密码子使用偏好对人溶菌酶基因翻译起始区前22位密码子（LYZop22）和全局密码子(LYZop)分别进行优化。构建人溶菌酶-荧光素酶融合表达载体（pGL3-LYZcw/op22/op）和人溶菌酶过表达载体（pcDNA-LYZcw/op22/op）,将上述载体分别转染牛乳腺上皮细胞(BMEC)、牛成纤维细胞（BFFC）和C127小鼠乳腺上皮细胞等3种细胞,通过荧光素酶、实时荧光定量PCR及Western-blot等方法检测密码子优化对溶菌酶表达的影响。【结果】牛乳蛋白基因密码子偏好以GC结尾,GC3s平均含量为0.537±0.062,而人溶菌酶GC3s含量为0.407,偏好以AT结尾;聚类结果表明,牛乳中酪蛋白与乳清蛋白类基因在密码子使用偏好性上也存在一定差异。依据筛选获得的牛主要乳蛋白基因5个高频密码子（RSCU>1.5）和7个低频密码子（RSCU<0.5）对人溶菌酶基因密码子进行优化并转染多种细胞进行表达效果分析。荧光素酶分析发现,相比野生型溶菌酶密码子（LYZcw）,翻译起始区密码子优化类型LYZop22分别在BMEC、BFFC细胞中提高1.48倍（P<0.01）和1.30倍（P>0.05）;而全局密码子优化类型LYZop分别在BMEC、BFFC中提高2.2倍（P<0.01）和2.44倍（P<0.01）,说明密码子优化能明显提高人溶菌酶在多种细胞中表达量。实时荧光定量PCR结果表明,LYZop22相比LYZcw在BMEC和BFFC细胞中分别提高了2.08倍（P<0.05）和1.5倍（P>0.05）,而LYZop则分别提高了22倍（P<0.01）和17.8倍（P<0.01）,mRNA表达水平与密码子优化后的mRNA二级结构稳定性呈正相关。Western-blot结果也进一步表明,密码子优化后的LYZop22和LYZop能明显提高重组人溶菌酶在牛乳腺上皮细胞中的表达量。上述结果表明,根据牛乳腺中主要乳蛋白基因密码子使用偏好进行密码子优化,能显著提高人溶菌酶在牛乳腺上皮细胞及成纤维细胞中的表达量,且溶菌酶基因全局密码子优化效果优于翻译起始区密码子优化效果。【结论】通过生物信息学分析获得了牛乳蛋白基因使密码子使用偏好及高低频密码子;依据牛乳蛋白密码子使用偏好性对人溶菌酶密码子优化能显著提高重组人溶菌酶mRNA水平和蛋白水平的表达量,为今后利用生物反应器高效生产重组人溶菌酶奠定基础。     

Achyranthes aspera (Prickly chaff flower) leaves- and seeds-supplemented diets regulate growth,innate immunity,and oxidative stress in Aeromonas hydrophila-challenged Labeo rohita

ABSTRACT

The immunostimulatory and disease-resistance properties of Achyranthes aspera were evaluated in rohu (Labeo rohita) challenged with Aeromonas hydrophila. Experimental diets were enriched with leaves at 0.25% (D1) and 0.5% (D2) and seeds at 0.5% (D3); the control diet (D4) was without any enrichment. Rohu (2.02 ± 0.23 g) were cultured for 75 days and then challenged with bacteria. The highest average weight was observed in the D3 diet-fed fish. The cumulative mortality rates were 70%, 60%, 40%, and 30% in the D4, D1, D2, and D3 diets fed to rohu respectively. Enriched diets significantly increased myeloperoxidase, nitric oxide synthase, and serum lysozyme levels and decreased malondialdehyde and carbonyl protein content. Expressions of lysozyme C and lysozyme G were significantly (P < .05) higher in the D3 diet-fed fish. In the kidney, IL-1β and TLR 4 were up-regulated in enriched-diet-fed fish. Supplementation of seeds and leaves at 0.5% showed a positive impact in fish.     

Effect of dietary GroBiotic®‐A supplementation as a prebiotic on the intestinal microflora,growth performance,haemato‐serological parameters,survival rate and body composition in juvenile beluga (Huso huso Linnaeus, 1754)

The objectives of the present study were to investigate of GroBiotic^®‐A (GBA) on growth, autochthonous intestinal microbiota and haemato‐serological parameters of beluga juvenile. A total of 180 fish (40.82 ± 5.81 g) were fed diets containing graded levels of GBA (0, 5, 10 and 20 g kg^?1 diet) for 8 weeks. No significantly differences in body composition, total viable aerobic bacteria, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, neutrophils, eosinophils, monocytes, albumin, glucose, triglyceride, cholesterol, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and lactate dehydrogenase were detected between treatment groups. The fish fed on 10 and 20 g kg^?1 GBA significantly showed higher lactic acid bacteria, final weight, body weight increases, weight gain and lower feed conversion ratio compared with the control and 5 g kg^?1 groups. The group fed 20 g kg^?1 GBA showed a highly significant difference in condition factor, survival rate, final length, total red and white blood cells, lymphocytes, haematocrit, haemoglobin, total protein, total immunoglobulin and lysozyme activity. The specific growth rate of the treatment groups was significantly elevated compared with the control groups. These results indicated that GBA at level 20 g kg^?1 improved growth, welfare and survival of beluga juvenile.     

Effect of oregano (Origanum onites L.) essential oil on growth,lysozyme and antioxidant activity and resistance against Lactococcus garvieae in rainbow trout,Oncorhynchus mykiss (Walbaum)

The aim of this study was to investigate the effects of different levels of Origanum onites L. essential oil as feed additives on the growth performance, antioxidant activity and disease resistance of rainbow trout. Fish (26.05 ± 0.15 g) were fed the experimental diets supplemented with four different concentrations (0.125, 1.5, 2.5 and 3.0 mL kg^?1) of O. onites essential oil for 90 days. Fish fed diets containing essential oil of O. onites had significantly higher final weight than the control group. Feed conversion ratio in fish fed diets containing 1.5 and 3.0 mL kg^?1 essential oil of O. onites was improved than other treatments (P < 0.05). The lowest feed conversion efficiency ratio was recorded in the 0.125 mL kg^?1 group of O. onites. Antioxidant status of fish was assayed for levels of plasma superoxide dismutase (SOD) and plasma catalase (CAT) activity. Lysozyme activity in plasma was significantly higher in fish fed diet containing 3.0 mL kg^?1 essential oil of O. onites (P < 0.05). After 8 weeks of feeding, fish were challenged with Lactococcus garvieae and cumulative mortality was recorded over 15 days. Dietary administration of 0.125, 1.5 and 2.5 mL kg^?1 O. onites significantly reduced fish mortality (P < 0.05). The 3.0 mL kg^?1 diet showed no mortality after challenged with L. garvieae. These results suggested that the essential oil of O. onites could be applied as growth promoter and also improved disease resistance when added to rainbow trout feed.     

溶菌酶对犊牛腹泻和生长性能的影响

[目的]本试验研究溶菌酶在使用量不同情况下,对犊牛腹泻和生长性能的影响。[方法]选择无病、健康、体型近似的犊牛100 头,随机分为4 组,每组25 头,分别为对照组、低剂量组（Ⅰ组、1 g/头·天）、中剂量组（Ⅱ组、3 g/头·天）、高剂量组（Ⅲ组、5 g/头·天）,试验期30 天。在试验开始和结束时,观察犊牛粪便、腹泻、体重、体尺变化,并记录。[结果]试验期溶菌酶对犊牛的粪便评分有影响,1~30 天的粪便评分Ⅲ组显著低于对照组（P<0.05）,且Ⅲ组比对照组降低12.5%;1~15 天时腹泻率差异显著（P<0.05）,Ⅰ组、Ⅱ组、Ⅲ组与对照组相比分别降低23.02%、41.81%、53.64%,表明溶菌酶使用量多,腹泻效果更加明显;添加溶菌酶的Ⅱ组和Ⅲ组犊牛的平均日增重均显著高于对照组（P<0.05）,分别提高9.6%、12.0%;Ⅲ组胸围显著高于对照组,高3.0%。[结论]溶菌酶使用到合适剂量,不仅能降低犊牛腹泻率,还能促进犊牛的生长性能。     

Evaluation of dietary chitosan effects on growth performance,immunity, body composition and histopathology of Nile tilapia (Oreochromis niloticus) as well as the resistance to Streptococcus agalactiae infection

The present investigation aimed to evaluate the effect of dietary chitosan supplementation on growth performance, body composition, immune response and histopathology of Nile tilapia, and also the in vitro antibacterial activity of chitosan against Streptococcus agalactiae (S. agalactiae). About 180 fish (average body weight 39.3 ± 0.3 g) were randomly divided into three groups according to chitosan supplementation: control group (basal diet without chitosan), Ch3 group (3 g chitosan/kg diet) and Ch5 group (5 g chitosan/kg diet). Growth performance parameters and body proximate composition were measured before infection but biochemical parameters and lysozyme and antibacterial activities before and after experimental infection. Results of the present investigation showed dietary chitosan (5 g chitosan/kg diet) significantly (p < .05) improved growth performance parameters, body composition (dry matter, crude protein, ether extract, ash, and carbohydrate) and serum biochemistry (total protein, albumin, globulin, with no effect on AST, ALT, urea and creatinine) before infection in Ch5 group than the control. After infection, liver enzymes (serum AST and ALT) were maintained lower in fish fed Ch3 or Ch5 than the control. Serum lysozyme and bactericidal activities significantly increased (p < .05) in chitosan groups before and after the challenge. The mortality rate was markedly reduced in the Ch3 group and prohibited in the Ch5 group after the experimental infection. In conclusion, feeding 3 or 5 g chitosan/kg diet increased the growth rate and improved FCR of Nile tilapia. In addition, it reduced mortality by its antibacterial and immunostimulant effects.     

Effects of different levels of plant proteins on the ongrowing of meagre (Argyrosomus regius) juveniles at low temperatures

Four experimental diets with different inclusion levels of plant proteins and fish protein hydrolysates were compared with a commercial diet for meagre (Argyrosomus regius) ongrowing at optimal and suboptimal water temperature. Results in terms of growth in length and weight, conversion efficiency, dietary feed intake and utilization, body composition (whole fish and liver) as well as enzyme and immunological activities are presented. Fish growth was significantly reduced by the inclusion of plant proteins, although further addition of fish protein hydrolysates improved the results. Daily feed intake was not affected by plant protein inclusion in the diets, although the group fed the highest inclusion level showed lower ingestion than the rest of the groups, probably as a consequence of a reduced dietary palatability. The decrease in water temperature during the second part of the experiment had a negative effect on feed intake and fish growth. Gross visceral morphology of meagre fed the experimental diets was not affected, but muscle weight was significantly reduced. Whole body and liver composition was not affected with plant protein inclusion. However, the inclusion of fish protein hydrolysates resulted in a significant increase in fat content, especially in liver cholesterol and steryl esters, with a parallel reduction in protein. Brush border enzymes were affected by plant protein inclusion as well as serum lysozyme that significantly increased in the fish fed the highest inclusion level. As a conclusion, up to 315 g kg^?1 plant protein (76.2% of total protein content) can be included in the diet for meagre without affecting growth or feed utilization. Higher inclusion levels can also be used if at least 5% fish protein hydrolysate is also included.     

高活力蛋清溶菌酶制备技术的研究

溶菌酶是一种无毒、无害、安全性较高的高盐基水解蛋白酶,在食品和医药领域有着广泛的用途。试验以蛋清为原料,通过阳离子交换树脂法提取高活力(纯度)溶菌酶。试验确定吸附与洗脱条件,蛋清与树脂的体积比为5∶1,搅拌时间为3.5h,洗脱液的离子强度为1mol/L,pH值为8.0,洗脱液经等电点沉淀和透析进一步分离纯化,所得浓缩液以110℃的进风温度和70℃的出风温度进行喷雾干燥得溶菌酶,产品活力为20650U/mg,水分含量为6.2%。     

溶菌酶在食品工业中的应用

综述了溶茵酶的基本性质,着重讨论了溶菌酶抑制的微生物种类、机理,系统地阐述了溶菌酶的制备及其在食品工业中的应用进展.     

牛乳溶菌酶概述及应用

溶菌酶又称胞壁质酶,广泛存在于自然界的动物、植物及微生物中。具有抗菌消炎、抗病毒、增强免疫力以及促进组织修复等生物学特性。并且应用于食品、饲料、医学及生物工程之中。针对目前临床中,对于奶牛乳房炎广泛大量使用抗生素,并且存在耐药性、药物残留等现象。重组溶菌酶质粒可以作为基因工程药物替代抗生素对乳房炎的防治,并且安全、无残留、无副作用。同时也还有待于进一步的研发和推广。