植物甜蛋白monellin基因工程菌表达条件的研究

将带有monellin基因的重组分泌型表达载体pETMO转入大肠杆菌BL21(DE3),得到重组工程菌株BMC33。经IPTG诱导,其所含有的甜蛋白基因可高效表达。研究不同的表达条件对甜蛋白表达水平的影响,得到优化发酵条件:装液量为50 mL/250 mL三角瓶,当培养液OD值达0.8时,添加诱导剂IPTG至终浓为0.9mmol/L,32℃诱导5 h。此时,甜蛋白monellin表达量可高达细菌可溶性蛋白的44.8%。     

甜蛋白monellin基因在酿酒酵母中的分泌表达

人工合成的单链甜蛋白monellin基因与经改造后的基因分别克隆到带有酵母半乳糖可诱导启动子GAL1的穿梭表达质粒pYES2.0中,得到表达载体pYESM及pYESMT。在表达载体pYESMT中,monellin基因的上游连接酿酒酵母的α-信号肽序列,得到分泌表达载体pYESMTA。分别将3个重组质粒转化酿酒酵母菌株INVsc1中进行表达。具有突变monellin基因的菌株INVMT/pYESMT的monellin蛋白表达量明显高于含monellin基因的菌株。而含有pYESMTA的菌株可将monellin基因分泌到胞外,表明α-信号肽序列能很好地将酿酒酵母中的重组monellin蛋白引导到胞外,完成分泌表达,且表达产物具有生物活性。     

Monellin-EGFP 融合蛋白在毕赤酵母中的表达

根据已报道的Monellin甜蛋白氨基酸序列,结合毕赤酵母与桑树密码子的偏好性,人工设计合成单链monellin基因,并与增强型绿色荧光蛋白EGFP基因连接,构建融合表达载体。采用电击转化法成功将表达载体导入毕赤酵母GS115中,并获得高效表达的重组子pPIC9K-M-E。经PCR检测,SDS-PAGE和Western Blot杂交证实已表达出融合蛋白,该蛋白经纯化与盲测实验,结果显示其甜度约为标准蔗糖的500倍。     

透明颤菌血红蛋白基因（vgb）与monellin基因在毕赤酵母中的联合表达

根据酵母偏爱密码子合成了monellin甜蛋白基因和vgb基因,然后构建了2种不同方式表达的载体,胞内表达的pPIC3.5KV和分泌表达的pPIC9KM.电击转化得到重组子GS115/pPIC9KM和GS115/pPIC9KM/pPIC3.5KV,通过PCR、SDS.PAGE及Western blotting检测发现monellin甜蛋白基因和vgb因成功整合进毕赤酵母基因组并成功表达,通过品尝和CO-差示光谱法证实其表达产物具有活性,用southern blotting对6株高产菌株进行拷贝数分析并未发现拷贝数与分泌蛋白产量之间存在明显的正相关.对比发现,含vgb基因菌株比未含菌株高甜度monellin的产量最高提高了20%,菌体密度平均提高了9%.     

马槟榔甜蛋白基因(MabinlinⅡ)的密码子优化及其在大肠杆菌中的表达

本研究根据大肠杆菌密码子的偏好性,对马槟榔甜蛋白基因MabinlinⅡ(A链+B链)的密码子进行优化,并将优化后的马槟榔甜蛋白基因命名为MabinlinⅡ(AB)Plus。将MabinlinⅡ(AB)Plus基因与大肠杆菌表达载体pET-28a(+)片段连接,构建重组表达载体pET-28a(+)-MabinlinⅡ(AB)Plus,进而转化到大肠杆菌宿主表达菌株BL21(DE3),IPTG诱导蛋白表达。结果表明,密码子优化后的基因MabinlinⅡ(AB)Plus在大肠杆菌中最佳表达条件为:最适IPTG浓度1.4 mmol/L,最佳诱导剂温度为37℃,最适菌株起始生长量OD₆₀₀为0.7,最佳诱导时间8 h。在最佳诱导表达条件下,目的蛋白表达量占菌体总蛋白约33.0%,与优化前约有提高7%。目的蛋白的上清液经过亲和纯化,在19 kDa处有单一的目的条带。经过感官评定,目的蛋白的甜度约为2%标准蔗糖溶液的400倍。这为研发以马槟榔甜蛋白为基础的新型甜味剂提供了科学的理论依据。     

地衣芽孢杆菌耐高温α-淀粉酶基因在大肠杆菌中的克隆及表达

用PCR方法从地衣芽孢杆菌中扩增了耐高温α淀粉酶基因,将扩增的DNA片段插入到大肠杆菌载体pUC19中,构建重组分泌型表达载体pUAM。pUAM中的耐高温α淀粉酶基因在大肠杆菌JM109中得到表达。经SDSPAGE分析显示,蛋白表达产物的分子量为55ku,同核酸序列测定所推导的值相符。     

牛凝乳酶原基因在大肠杆菌中的高效表达及活性检测

以实验室保存的携带凝乳酶原前体基因的重组载体pMD 19-T/bPPC为模板克隆凝乳酶原基因,经双酶切后与载体pET-30a连接得到重组载体pET-30a/bPC,转化大肠杆菌BL21(DE3),经IPTG诱导后,采用SDS-PAGE检测目的蛋白表达情况。重组蛋白经变性/复性、DEAE-Sepharose Fast Flow纯化和自催化后检测凝乳活性。结果表明,重组凝乳酶原基因在大肠杆菌中高效表达,表达量占菌体总蛋白的68%,采用Arima K方法检测,其凝乳活力达到80 SU/mL。因此,通过大肠杆菌表达系统大量制备具有生物活性的重组牛凝乳酶原的策略是可行的,研究结果为弥补国内天然牛凝乳酶的短缺提供一种途径。     

甜荞麦16 kDa过敏原基因的克隆及其原核表达载体的构建

目的 对甜荞麦(common buckwheat)过敏原的分子生物学开展研究。方法 通过RT-PCR克隆甜荞麦16 kDa过敏原蛋白的全长基因, 并根据序列设计带有酶切位点的特异性引物, 扩增甜荞麦16 kDa过敏原基因的完整开放阅读框, 与pET-28a载体连接, 构建原核表达载体。 结果 本研究成功克隆了甜荞麦16 kDa过敏原蛋白的基因, 且构建了其原核表达载体。该基因含有长度为450 bp的开放阅读框, 编码149个氨基酸, 在GenBank数据库中的登录号为EU883600, 同源性分析发现其与数据库中已知的荞麦过敏原基因有高度的同源性。结论 本研究为甜荞麦16 kDa过敏原蛋白的重组表达和临床过敏性疾病的诊断奠定了基础。     

植物乳杆菌亚硝酸盐还原酶基因在大肠杆菌中表达

构建乳杆菌亚硝酸盐还原酶基因的重组质粒并表达。以植物乳杆菌基因组DNA为模板,PCR扩增亚硝酸盐还原酶基因,连接到表达载体pET-32a(+)上,构建的重组载体转入大肠杆菌BL21(DE3)中,经IPTG诱导表达后,菌体总蛋白经SDS-PAGE鉴定显示重组质粒诱导表达产物有特异性蛋白条带。该表达产物经细菌亚硝酸盐还原酶试剂盒测定显示有明显的酶活性。实验结果为深入研究植物乳杆菌亚硝酸盐还原酶提供有益的参考。     

采用狂犬病毒G基因原核表达的间接ELISA方法

为获得狂犬病毒G基因并在大肠杆菌中进行表达,进而初步建立用于评价狂犬疫苗免疫效果的方法,根据狂犬病病毒RV(rabies virus)ERA株G基因序列设计引物,从病毒中提取出RNA,经逆转录并扩增得到RVG基因;将该基因片段定向克隆到原核表达载体pET28a(+)中,构建重组质粒PET-RVG并转化到大肠杆菌BL21(DE3)gold中,经IPTG诱导表达并确定表达的最佳条件;Ni亲和层析柱纯化获得重组G蛋白,并以G蛋白为抗原建立检测狂犬病毒抗体的间接ELISA方法.检测结果表明,经灰度分析纯化后纯度可达96%.     

High pressure inactivation of Escherichia coli, Campylobacter jejuni, and spoilage microbiota on poultry meat

This study evaluated the high pressure inactivation of Campylobacter jejuni, Escherichia coli, and poultry meat spoilage organisms. All treatments were performed in aseptically prepared minced poultry meat. Treatment of 19 strains of C. jejuni at 300 MPa and 30°C revealed a large variation of pressure resistance. The recovery of pressure-induced sublethally injured C. jejuni depended on the availability of iron. The addition of iron content to enumeration media was required for resuscitation of sublethally injured cells. Survival of C. jejuni during storage of refrigerated poultry meat was analyzed in fresh and pressuretreated poultry meat, and in the presence or absence of spoilage microbiota. The presence of spoilage microbiota did not significantly influence the survival of C. jejuni. Pressure treatment at 400 MPa and 40°C reduced cell counts of Brochothrix thermosphacta, Carnobacterium divergens, C. jejuni, and Pseudomonas fluorescens to levels below the detection limit. Cell counts of E. coli AW1.7, however, were reduced by only 3.5 log (CFU/g) and remained stable during subsequent refrigerated storage. The resistance to treatment at 600 MPa and 40°C of E. coli AW1.7 was compared with Salmonella enterica, Shiga toxin-producing E. coli and nonpathogenic E. coli strains, and Staphylococcus spp. Cell counts of all organisms except E. coli AW 1.7 were reduced by more than 6 log CFU/g. Cell counts of E. coli AW1.7 were reduced by 4.5 log CFU/g only. Moreover, the ability of E. coli AW1.7 to resist pressure was comparable to the pressure-resistant mutant E. coli LMM1030. Our results indicate that preservation of fresh meat requires a combination of high pressure with high temperature (40 to 60°C) or other antimicrobial hurdles.     

响应面法优化酿酒酵母工程菌产甜蛋白monellin发酵培养基

对酿酒酵母工程菌WHF9/monellin在液体培养基中产甜蛋白monellin的条件进行了优化.采用单因子试验筛选出细胞生长培养基的最适碳源为蔗糖,氮源为玉米浆干粉,且应添加微量元素溶液.在此基础上,利用Plackett-Burman试验设计筛选出影响菌体生物量的3个显著因素:蔗糖、玉米浆干粉、微量元素溶液.用最陡爬坡路径逼近最大响应区域后,利用Box-Behnken设计和响应面分析法对显著因素进行优化,得出蔗糖、玉米浆干粉、微量元素溶液的最佳浓度分别为102.2g/L,33.9g/L,4.5mL/L.菌株在优化后的生长培养基中的生物量比在YPD培养基中提高了81.38％.在诱导剂半乳糖最适浓度102.2g/L条件下,菌株在优化后的培养基中monellin的表达量达到226.7mg/L,比在YPG中表达的monellin提高了4.2倍,且表达的monellin具有生物活性.     

Evaluation of a conductance method for enumerating Escherichia coli on chicken, pork, fish, beef, and milk

Experiments were conducted to evaluate a rapid method for enumerating Escherichia coli on food products of animal origin. In study I, rinses from samples of chicken, ground beef, pork, and fish and samples of milk were inoculated with various levels of actively growing E. coli. Conductance assays were monitored at 44 degrees C on each sample using coliform medium supplemented with 2% dextrose. High correlations between E. coli concentrations and E. coli conductance detection times (ECDTs) were found (r = -0.97 to -0.99) for all foods tested in all replicates; however, in most cases, the concordance correlation coefficients (r(c)) were low, indicating a lack of predictive accuracy. In this study, low accuracy of the conductance method for estimating E. coli counts was attributed to use of concentrations of E. coli that exceed 10(6) CFU/ml, the detection threshold of the instrument. Slopes of the linear regression lines (E. coli concentration vs. ECDT) for each type of food tested were not significantly different (P < 0.0001), indicating that a single regression equation may be used to estimate E. coli counts for all of the types of food tested in 1 to 7.5 hours using ECDT. In study II, ECDTs for pork, fish, beef, and milk significantly (P < 0.05) decreased in a linear manner as time of temperature abuse increased. Although the ECDT for chicken decreased linearly, no significant differences were observed between 3 and 6 or between 9 and 12 h of abuse. These data demonstrate a strong relationship between increasing populations of E. coli due to temperature abuse and decreasing ECDT. Therefore, results from both studies indicate that this method could be useful for estimating naturally occurring populations of E. coli on foods of animal origin.     

Comparison of rapid enzyme-linked immunosorbent assay and immunomagnetic separation methods for detection of Escherichia coli O157 in fecal, hide, carcass, and ground beef samples

Rapid enzyme-linked immunosorbent assays (ELISAs) are approved for detection of Escherichia coil O157 in beef products. However, these kits have also been used in the industry to detect this pathogen on hides or in feces of cattle, although this use has not been validated. The objective of this study was to compare commercially available ELISAs (E. coli Now, Reveal, and VIP) with immunomagnetic separation along with selective media to detect E. coli O157 on hides, in feces, and in medium- and low-level-inoculated ground beef and carcasses (simulated by using briskets) samples. Naturally infected hide and fecal samples were subjected to both the immunomagnetic separation method and ELISAs for the detection of E. coli O157. Additionally, E. coli O157 inoculated and noninoculated ground beef and beef briskets were used to simulate meat and carcass samples. When comparing the detection results from the ELISAs (E. coli Now, Reveal, and VIP) to the immunomagnetic separation method, poor agreement was observed for fecal samples (kappa = 0.10, 0.02, and 0.03 for E. coli Now, Reveal, and VIP, respectively), and fair-to-moderate agreement was observed for hide samples (kappa = 0.30, 0.51, and 0.29 for E. coli Now, Reveal, and VIP, respectively). However, there was near-perfect agreement between the immunomagnetic separation method and ELISAs for ground beef (kappa = 1, 1, and 0.80 for E. coli Now, Reveal, and VIP, respectively) and brisket (kappa = 1, 1, and 1 for E. coli Now, Reveal, and VIP, respectively) samples. Assuming immunomagnetic separation is the best available method, these data suggest that the ELISAs are not useful in detecting E. coli O157 from hide or fecal samples. However, when ELISAs are used on ground beef and beef brisket samples they can be used with a high degree of confidence.     

Acid stress, starvation, and cold stress affect poststress behavior of Escherichia coli O157:H7 and nonpathogenic Escherichia coli.

The effects of acid shock, acid adaptation, starvation, and cold stress of Escherichia coli O157:H7 (ATCC 43895), an rpoS mutant (FRIK 816-3), and nonpathogenic E. coli (ATCC 25922) on poststress heat resistance and freeze-thaw resistance were investigated. Following stress, heat tolerance at 56 degrees C and freeze-thaw resistance at -20 to 21 degrees C were determined. Heat and freeze-thaw resistance of E. coli O157:H7 and nonpathogenic E. coli was enhanced after acid adaptation and starvation. Following cold stress, heat resistance of E. coli O157:H7 and nonpathogenic E. coli was decreased, while freeze-thaw resistance was increased. Heat and freeze-thaw resistance of the rpoS mutant was enhanced only after acid adaptation. Increased or decreased tolerance of acid-adapted, starved, or cold-stressed E. coli O157:H7 cells to heat or freeze-thaw processes should be considered when processing minimally processed or extended shelf-life foods.     

食源性大肠杆菌O157毒力、耐药性及CRISPR分型分析

为深入了解食品源大肠杆菌O157的生物学特点,本研究对2014-2015年分离的39株大肠杆菌O157进行了PCR鉴定,毒力基因和耐药性检测,并利用规律成簇的间隔短回文重复序列(CRISPR)分型技术对菌株的遗传特性进行了分析。结果表明,39株菌株中有8株鉴定为O157:H7,31株为O157;检测的11种毒力基因中,eae存在于所有菌株中,stx2存在于82.50%菌株中,stx1未检出,其他毒力岛基因esp A、etp D、tir、tox B、iha和kat P携带率分别为92.31%、94.87%、87.18%、79.49%、69.23%和46.15%。药敏试验结果表明,菌株对四环素、复方新诺明、链霉素、氯霉素和氨苄西林等抗生素高度耐药,超过30%菌株具有多重耐药性。CRISPR分型结果表明菌株具有较高的遗传多样性,39株菌株中有33株存在CRISPR1位点,8株E.coli O157:H7产生了相同的CRISPR spacer图谱,而26株E.coli O157产生了13种spacer图谱。本研究为食源性疾病的监测、疾病溯源和流行病学研究提供重要的基础数据。     

Evaluation of nonpathogenic surrogate bacteria as process validation indicators for Salmonella enterica for selected antimicrobial treatments, cold storage, and fermentation in meat

Prerigor lean and adipose beef carcass tissues were artificially inoculated individually with stationary-phase cultures of five nonpathogenic Escherichia coli cultures that had been previously identified as surrogates for E. coli O157:H7 or a mixture of five Salmonella strains in a fecal inoculum. Each tissue sample was processed with microbial interventions comparable with those used in the meat industry. The log reductions of the E. coli isolates were generally not statistically different from the salmonellae inoculum within a specific treatment. Inoculation experiments were also conducted with ground beef stored at either 4 or -20 degrees C. When compared with the Salmonella inoculum, at least three of the five E. coli strains survived in a manner that was not statistically different from the salmonellae. The E. coli strains and the Salmonella mixed culture were also inoculated into summer sausage batter, and the population enumerated both before and after fermentation. Four of the E. coli strains showed a lower population reduction (higher survival) than the Salmonella mixed culture. The five nonpathogenic E. coli strains may be used as individually or collectively for specific process validation indicators for Salmonella.