产纤溶酶菌株的分离和鉴定及纤溶酶基因在酿酒酵母中的表达

目的:从豆豉中分离具有强纤溶活性的菌株,实现纤溶酶基因在酿酒酵母中的表达.方法:通过纤维蛋白平板法从豆豉中分离具有强纤溶活性的菌株,结合形态特征、生理生化特性和16S rDNA序列分析鉴定该菌株;通过PCR扩增豆豉纤溶酶基因,构建pYES2-DFE重组质粒,转化酿酒酵母H158感受态细胞,经β-半乳糖诱导表达后,用纤雏蛋白平板法分别检测发酵液上清和菌体破碎上清的纤溶酶活性.结果:鉴定该菌株为枯草芽孢杆菌(Bacillus subtilis);活性检测结果是酿酒酵母发酵液上清具有纤溶酶活性,而菌体破碎上清没有活性.结论:豆豉纤溶酶基因在酿酒酵母中实现了分泌表达.     

纤溶活性重组纳豆激酶在大肠杆菌中的表达

通过PCR方法扩增纳豆激酶成熟肽和纳豆激酶酶原(proNK)基因片段,并分别克隆到表达载体pET28a上,构建与His标签融合表达的重组表达质粒pET28a-NK和pET28a-proNK,转化大肠杆菌宿主菌BL21(DE3),IPTG诱导表达,SDS-PAGE电泳和纤维蛋白平板检测目的蛋白的表达及表达产物的纤溶活性.结果表明,纳豆激酶成熟肽基因在大肠杆菌中的表达产物以包涵体形式存在,表达产物没有纤溶活性;纳豆激酶酶原基因在大肠杆菌中的表达产物能自我剪切,形成有纤溶活性的纳豆激酶,但同时对宿主细胞有降解毒性.     

纳豆激酶基因在酿酒酵母中的表达

目的:从纳豆芽孢杆菌基因组DNA中扩增纳豆激酶基因,以实现该基因在酿酒酵母中的表达.方法:通过PCR方法扩增纳豆激酶基因,利用DNA重组技术构建重组质粒pYES2-NK,转化酿酒酵母H158感受态细胞;经β-半乳糖诱导表达后,用纤维蛋白平板法检测纤溶酶活性.结果:克隆到大小为1192bp的纳豆激酶基因,编码397个氨基酸;活性检测表明酿酒酵母发酵液的上清液具有纤溶酶活性.结论:纳豆激酶基因在重组酿酒酵母中实现了分泌表达.     

基于分光光度法构建纳豆激酶纤溶活性快速测定方法

为建立一种快捷、有效的纳豆激酶纤溶活性测定方法,在研究纤维蛋白平板法的基础上,构建了以透明圈面积为中间参数的纳豆激酶纤溶活力与吸光度值之间关系模型,通过测定纳豆激酶显色反应中的吸光度值,直接获得纳豆激酶的纤溶活性。结果显示改进后的酪蛋白方法可行,其模型方程为y=1877.77x-481.08（R2=0.9924）。验证结果显示模型相关性强,准确度较高,操作简便、快速、检测成本低,是一种高效的纳豆激酶纤溶活性快速检测方法。      

鸡卵黄前体物和受体及其对蛋品品质的影响

鸡卵黄前体物,即极低密度脂蛋白(VLDL)和卵黄生成素(VTG),是由性成熟母鸡分泌雌激素刺激肝脏而合成的,主要影响鸡蛋的内在品质(脂肪酸和胆固醇等)。极低密度脂蛋白受体(VLDLR)是卵黄前体物的转运受体,介导VLDL和VTG从卵黄膜表面进入卵母细胞并促进卵母细胞的成熟,是影响产蛋数和卵黄质量的关键基因。因此,鸡卵黄前体物及其受体与蛋鸡产蛋性能及蛋品品质密切相关。本文综述鸡卵黄前体物及其受体的结构、功能以及它们对蛋品品质的影响。     

鸡胚营养组合物对衰老大鼠基因损伤修复的影响

目的:观察鸡胚营养组合物对衰老大鼠基因损伤修复的影响,探讨其延缓衰老的可能机制。方法:采用D-半乳糖建立衰老SD大鼠动物模型,每日1次颈背部皮下注射D-半乳糖500mg/(kg·d),连续造模90天,对照组颈背部皮下注射等剂量的生理盐水注射液。从第1天起,鸡胚营养组合物组、鸡胚组、营养组合物组、蛋清组,分别以相应剂量对大鼠进行灌胃,每天1次,直至第90天,拉颈处死大鼠。取各组大鼠肝脏、骨髓和脑组织,采用RT-PCR法检测各组织修复基因的表达情况。结果:与衰老模型组相比,鸡胚营养组合物组衰老大鼠肝脏和骨髓组织中错配修复基因MSH2表达量升高(P<0.05);鸡胚营养组合物组衰老大鼠肝脏组织中同源重组修复基因Xrcc1、Xrcc2、骨髓中同源重组修复基因Rad51以及脑组织中同源重组修复基因Rad51、Xrcc1表达量升高(P<0.05)。结论:鸡胚营养组合物能够修复衰老大鼠肝脏、骨髓和脑组织基因的损伤,此可能为其延缓衰老的作用机制之一。     

豆豉营养评价及其粗纤溶酶活性检测

试验检测了收集到的不同豆豉的蛋白质、水分、总糖含量等,了解各种豆豉的营养价值;并通过琼脂糖-纤维蛋白平板法测定了各种豆豉纤溶酶的活性,分析了影响豆豉纤溶酶粗酶液活性的因素。结果显示,豆豉样品DC-H8的营养价值较高;试验确定豆豉纤溶酶粗酶液的提取方法:取一定量的豆豉,保持豆豉整粒,加5倍体积的生理盐水,于4℃浸提16h,离心、过滤后测定酶活,豆豉样品DC-H8的纤溶酶活性最高,为285.759 IU/mL。     

抗变链球菌鸡卵黄免疫球蛋白(IgY)的制备及活性检测

以远缘链球菌(S.sorbrinus 6715血清型g)免疫产卵母鸡,应用酶联免疫吸附测定法(ELISA)测定鸡卵黄中免疫球蛋白(Immunoglobulin of Yolk,IgY)的活性,研究鸡免疫应答并制备出高免抗-S.sorbrinus IgY.结果显示以109 cfu/只剂量免疫的母鸡,第一次免疫后的第七天卵黄中出现特异性IgY,加强免设后抗体效价上升更快,经ELISA法检测,卵黄中的抗体效价到1384.而冷冻干燥后的IgY,其效价要超过12000.     

纳豆激酶原基因在毕赤酵母中的分泌表达

为构建纳豆激酶原基因的重组酵母菌分泌型表达载体pPRONK2,并在毕赤酵母菌中进行表达,将来源于豆豉的纳豆芽孢杆菌的纳豆激酶原基因克隆至毕赤酵母菌分泌型表达载体pHBM905A上,得到重组质粒pPRONK2,再将其经SalI酶切后分别转化3株毕赤酵母菌KM71、GS1 15、SMD1 168,在MD平板上筛选得到重组酵母菌.经检测重组酵母菌发酵液中具纳豆激酶纤溶活性.表明在毕赤酵母菌中成功地实现了纳豆激酶的分泌表达.     

纤溶酶的分离纯化及其胞外存在方式的初步探索

为了获得纯纤溶酶并确定纤溶酶在枯草杆菌DC-12胞外是否存在酶与酶原2种形式,收集枯草杆菌DC-12不同时间点的发酵上清液进行分离纯化,对纤溶酶及其可能存在的酶原进行纯化和分析,不同时间点的发酵液通过Sephadex G-75凝胶过滤,都出现2个吸收峰.层析液和加入纤溶酶原激活剂尿激酶的层析液在加热的纤维蛋白平板上都有水解圈,并且10μL层析液和2μL巴比妥那缓冲液在加热的纤维蛋白平板上产生的水解圈与10μL层析液和2μL 100U/mL尿激酶所产生水解圈的面积相同.通过SDS-PAGE凝胶电泳发现,Ⅰ峰在44.3ku～66.4ku处有一条带,Ⅱ峰在29ku处有一条带,表明获得了电泳纯的豆豉纤溶酶,可用于后续研究,初步判定豆豉纤溶酶在枯草杆菌DC-12胞外不存在酶原.     

禽蛋黄过敏原Gal d 6的重组表达、纯化鉴定

目的:建立表达蛋黄Gal d 6 重组蛋白工程菌,并鉴定重组Gal d6蛋白免疫活性。方法:直接合成Gal d 6 DNA,与pET-28a构建重组质粒,转化E.coli BL21经IPTG诱导表达;优化表达条件制备重组Gal d 6蛋白,经镍离子亲和层析柱纯化得到纯品蛋白;纯化Gal d6蛋白经间接ELISA及western blot鉴定Gal d 6蛋白免疫活性。结果:经DNA测序、SDS-PAGE、禽蛋过敏血清鉴定表达Gal d 6蛋白具有免疫活性,经镍柱亲和层析获得单一条带。该工程菌最佳表达条件为37 ℃,0.5 mmol/L IPTG,2 h,收获量每升菌液可收获重组蛋白约9.1 mg。重组蛋白与禽蛋过敏血清具有良好反应性。结论:建立稳定表达具有免疫活性的Gal d 6蛋白的工程菌株。     

腰果主要过敏原Ana o 2原核表达及免疫学鉴定

目的 构建Ana o 2重组表达质粒,原核表达重组蛋白并评价其免疫活性。方法 将Ana o 2基因构建到pET-28a(+)载体中,测序正确的重组质粒转化Rosetta(DE3)感受态细胞,诱导表达目的蛋白并进行质谱鉴定。利用腰果过敏阳性血清,通过酶联免疫吸附试验(ELISA)和蛋白质免疫印记(Western blot)法评价重组Ana o 2的免疫活性。结果 重组蛋白进行十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)表明分子量为54 kD,与理论值相符,经质谱鉴定为Ana o 2。ELISA结果显示,用重组Ana o 2检测腰果过敏阳性血清与阴性血清特异性IgE抗体(sIgE)水平差异有统计学意义(t=2.44,P<0.05)。Western blot结果表明,重组Ana o 2与腰果过敏患者血清反应性良好。结论 利用原核系统表达了Ana o 2,并且重组Ana o 2与腰果过敏患者血清具有良好的反应性。     

黄喉拟水龟蛋营养成分分析及评价

对黄喉拟水龟蛋的蛋白质、氨基酸、矿物质、蛋黄中磷脂及脂肪酸组成等营养成分及价值进行评价。结果表明:与鸡蛋的营养组成相比,黄喉拟水龟蛋蛋清蛋白质种类多于鸡蛋蛋清,氨基酸组成符合理想模式,铁、镁、锌、磷含量高。龟蛋黄脂肪含量低于鸡蛋蛋黄,脂肪酸种类多,不饱和脂肪酸占总脂肪酸的80.72%,多不饱和脂肪酸二十二碳六稀酸(docosahexaenoic acid,DHA)6.39%,花生四稀酸3.72%,二十碳五稀酸(eicosapentaenoic acid,EPA)3.69%。龟蛋蛋白质种类丰富,矿物质和不饱和脂肪酸含量高,是一种高价值优质健康食品。     

抗克百威单链抗体的可溶性表达载体的构建及鉴定

为构建克百威（carbofuran,CBF）单链抗体（sc Fv）可溶性表达载体,实现其在大肠杆菌中的表达。以p CANTAB5E-sc Fv质粒为模板扩增CBF的重链（VH）及轻链（VL）片段,通过引物设计引入由15个氨基酸组成的连接肽（Gly4Ser）3,经重叠延伸拼接重链及轻链片段,并通过PCR扩增得到sc Fv基因,再与载体p LIP6/GN连接,转化大肠杆菌BL21,阳性克隆质粒经PCR鉴定并测序。重组菌经0.6μmol/L异丙基硫代半乳糖苷（IPTG）及低温诱导表达重组单链抗体,并通过SDS-PAGE和Western blotting对其鉴定。采用ELISA法检测重组单链抗体的抗原结合活性。结果表明重组质粒p LIP6/GN-sc Fv含有插入片段,sc Fv与碱性磷酸酶（AP）相连得到重组蛋白的分子量接近78 ku,可被游离的CBF竞争性抑制,IC50值为26.80 ng/m L。这说明成功构建了重组质粒p LIP6/GN-sc Fv并实现了其在大肠杆菌中的可溶性表达,为研究其在免疫分析方法中的应用奠定了基础。      

钝齿棒杆菌ArgR蛋白的表达、纯化及其多聚体的形成

通过PCR技术从钝齿棒杆菌(Corynebacterium crenatum)AS1.542基因组中扩增得到长度为516bp的argR基因,将其连接到原核表达载体pET22b(+),并转化至大肠杆菌BL21(DE3)获得重组菌株BL21(DE3)/pET22b-argR,以异丙基-β-D-硫代吡喃半乳糖苷(IPTG)在33℃诱导8h实现高效可溶性表达,获得了一个分子质量为20kD的融合蛋白ArgR。用纯化后的目的蛋白免疫小鼠制备多克隆抗体,间接ELISA检测抗体效价高达1:160000。Western blot分析结果表明,L-精氨酸介导重组蛋白形成的多聚体,可与自制的鼠抗ArgR多克隆抗体进行特异性反应,与预期结果一致,表明ArgR通过与精氨酸共孵育形成了多聚体,该结果有助于采用凝胶阻滞方法对ArgR蛋白与钝齿棒杆菌精氨酸生物合成途径中各操作子的结合情况进行进一步研究。     

Determination of egg content in egg pasta by an indirect elisa procedure

Polyclonal antibodies have been raised against a highly purified egg yolk protein, which appears to be unaffected by thermal treatments and whose content is constant in eggs of different origin. The antibodies are able to bind specifically to the egg yolk protein and can be used for the quantification of the egg content in egg pasta. For this purpose an indirect ELISA procedure has been developed.     

Changes in activity during storage and characteristics of superoxide dismutase from hen eggs (<Emphasis Type="Italic">Gallus gallus domesticus</Emphasis>)

Hen eggs are one of the most popular food stuffs. Moreover, they can be the source of not only nutrients but also factors of biological origin, which may be used for food preservation and food additives. The aim of the study was to determine and describe the activity of superoxide dismutase isoforms (SOD; EC 1.15.1.1) in hen eggs (Gallus gallus domesticus). Our electrophoretic studies confirmed the presence of SOD isoenzyme bands with molecular weight of 14–15 and 50–70 kDa in egg yolk. By contrast, in egg white, we confirmed the presence of a protein with molecular weight of 13–14 and 50–55 kDa. Zymografic pattern confirmed the activity of SOD isoforms of the enzyme present in the egg yolk; however, it did not confirm enzyme activity in egg white (at the level of error of the used method). Study has also shown SOD activity during storage at 4 °C for 9 days in egg yolk and egg white. In start time, SOD activity in egg yolk is clearly different from a small activity in the protein (respectively, 90.5 ± 22.2 and 7.9 ± 3.9 U g⁻¹). It did not change during 6 days storage but between 6th and 9th day, it decreased significantly in egg yolk while remained low but unchanged in egg white. Present study confirmed the presence of SOD and its activity in hen egg yolk.     

Physicochemical and functional properties of Chinese soft-shell turtle (Pelodiscus sinensis) egg

The physicochemical properties of Chinese soft-shell turtle egg were characterized for functional use in the food industry. The egg yolk of un-fertilized soft-shell turtle eggs was separated and fractionated into granules and plasma. Then, the egg yolk, albumen, granules, and plasma were freeze-dried for further analysis. Results showed that the Chinese soft-shell turtle egg typically comprised 50% egg yolk, 34% albumen, and 16% shell in average. The egg yolk composed of 61% granules and 39% plasma. The granules contained most of the protein, while the plasma contained most of the lipid in egg yolk. The albumen contained about 26% ash on a dry weight basis. Lysozyme was the major component in turtle egg albumen. The protein solubility of egg yolk, granules, and plasma was affected by the changes in pH, while that of albumen remains constant. The emulsifying and foaming properties increased when the concentration increased for all samples. Both the yolk and the albumen in turtle egg exhibited better functional properties than those in normal hen egg. These physicochemical and functional properties of Chinese soft-shell turtle egg are fundamental and essential for future study and food applications.     

Hen Egg Yolk Prevents Bacterial Adherence: A Novel Function for a Familiar Food

ABSTRACT: The aim of this study was to evaluate the ability of unfractionated hen egg yolk to prevent the attachment of Salmonella typhimurium to murine intestinal epithelial cells in vitro. Inhibition of adherence was examined microscopically and by an ELISA. Both methods showed that egg yolk from immunized and unimmunized hens significantly reduced bacterial adherence. Optical density (OD) readings ± SD of 0.825 ± 0.016 for untreated bacteria were reduced to 0.335 ± 0.024 and 0.267 ± 0.021 for bacteria pretreated with egg yolk from immunized hens and unimmunized hens, respectively. Microscopic analysis showed that egg yolk from either immunized or unimmunized hens reduced bacterial adherence from 17 ± 2.7 bacteria per epithelial cell to less than 4 bacteria per epithelial cell. These results show that hen egg yolk significantly inhibits adherence of S. typhimurium to intestinal epithelial cells in vitro. Boosting of specific antibody levels does not enhance this antiadhesive effect, suggesting that egg yolk contains novel antiadhesive factors.