洗必泰MIC作用下大肠杆菌和金黄色葡萄球菌 SDS-PAGE图谱的变化

[目的]探讨在洗必泰MIC(最小抑菌浓度)状态下大肠杆菌(8099株)、金黄色葡萄球菌(ATCC6538株)菌体蛋白质SDS—PAGE(十二烷基磺酸钠—聚丙烯酰胺凝胶电泳)图谱的改变，为消毒剂抑菌机理的研究和从蛋白质组角度寻找抑菌靶蛋白提供参考。[方法]以消毒试验常用的大肠杆菌、金黄色葡萄球菌标准菌株为例，收集在洗必泰作用下的菌体，SDS溶解菌体蛋白，SDS—PAGE后，对比研究洗必泰MIC、低于MIC以及无洗必泰作用下的菌体蛋白质在SDS—PAGE图谱中所表现出来的差异，并对试验的有关影响因素进行了摸索。[结果]在洗必泰MIC下，金黄色葡萄球菌的四条蛋白带(分别位于31KD左右、31KD和43KD之间、43KD左右处和大于97．4KD处)发生明显变化；大肠杆菌的一条蛋白带(位于66．2KD与97．4KD之间)发生明显变化。[结论]菌体蛋白条带在SDS—PAGE图谱中所表现出的变深或变浅的现象，提示在洗必泰MIC下菌体蛋白正常表达量的增多或减少。     

外膜蛋白SDS—PAGE对空肠弯曲菌的分型研究

用SDS—PAGE技术检测分析不同来源的41株空肠弯曲菌外膜蛋白特征。根据外膜蛋白SDS—PAGE凝胶上六条蛋白带多少的组合和外膜蛋白主带分子量的大小可将其各分为七个和九个型；根据外膜蛋白SDS—PAGE定量聚类分析可将其分为七个型(以95%相似性为水准)．结合此三种方法可更细致地鉴别不同来源的菌株，表明外膜蛋白SDS—PAGE技术是鉴别分型细菌的有效方法．     

T7噬菌体衣壳蛋白IOA的表达、纯化及多克隆抗体的制备

目的表达纯化T7噬菌体衣壳蛋白10A,并免疫家兔得到抗10．4多克隆抗体．为研究噬菌体展示抗体技术,进而筛选致病微生物蛋白抗体奠定基础。方法37℃培养含有17噬菌体10A蛋白质粒的大肠杆菌BLT5615,用异丙基-β-D-硫代半乳糖苷直接诱导表达该蛋白;SDS．PAGE鉴定表达状态,盐酸胍变性一尿素复性纯化以包涵体形式存在的蛋白并免疫家兔：辛酸一硫酸铵沉淀法纯化多克隆抗体,间接ELISA测定效价,WesternBlot鉴定特异性。结果成功表达了T7噬菌体衣壳蛋白10A。SDS—PAGE显示所表达的蛋白相对分子质量约为37kDa。主要以包涵体形式存在：复性后SDS—PAGE分析获得单一条带蛋白。利用该蛋白免疫家兔,6次免疫后抗血清的效价均达到1：160003。纯化后的多克隆抗体可以和17噬菌体衣壳蛋白10A特异结合。结论成功表达、纯化了17噬菌体衣壳蛋白10A,并制备了其多克隆抗体。     

人重组内皮抑素基因的克隆与融合表达

目的构建人内皮抑素(endostatin)融合蛋白表达载体,并在大肠杆菌中表达。方法用反转录多聚酶链反应(RT—PCR)得到人内皮抑素全基因,连接PGEM—T载体,测序确认,定向克隆重组入pTRX表达载体,转化大肠杆菌B121(DE3),IPTG诱导表达。结果经SDS—PAGE分析,出现特异性蛋白质条带,大小与预期相符合。结论人内皮抑素基因在原核细胞表达载体pTRX中获得成功表达,为应用内皮抑素进行肿瘤的抗血管生成治疗奠定了基础。     

舍蝇抗菌肽的提取及其对肿瘤细胞生长的抑制作用

目的：研究含蝇抗菌肽的提取对肿瘤细胞生长的抑制作用；方法：对舍蝇幼虫采用针刺损伤及金黄色葡萄球菌感染诱导方法，并通过研磨、加热、阳离子交换柱层析及Sephadex G—50葡聚糖凝胶柱层析等步骤提取抗菌肽；结果：经SDS—PAGE电泳分离到2条带，分子量分别为4kD，9．6kD；结论：提取的舍蝇抗菌肽能有效地抑制人胃癌细胞MGC80—3和BGC—823，人乳腺癌细胞MCF—7及人肺癌细胞SPC—A—1的体外增殖，并且呈浓度依赖关系。     

水痘-带状疱疹病毒糖蛋白I基因在昆虫细胞中的表达及纯化

目的 将北京水痘-带状疱疹病毒(VZV)84-7株克隆糖蛋白I(gpI)基因在杆状病毒-昆虫细胞表达系统中表达,并对其表达产物进行纯化。方法 采用PCR方法从VZVDNA中扩增gpI全基因序列,并将其插入杆状病毒转移质粒pBacPAK9中,获得重组转移质粒pBacVZVgpI,对pBacVZVgpI中的插入基因进行测序。重组转移质粒与线性杆状病毒BacPAK6DNA(Bsu36Idigested)共转染Sf9昆虫细胞,获得重组病毒BacPAK-gpI。通过亲和层析纯化重组蛋白,并检测其抗原性。结果 PCR扩增得到gpI基因,测序结果表明克隆的外源基因正确。经SDS-聚丙烯酰胺凝胶电泳(SDS—PAGE)、免疫印迹(weotem-blot)方法证明gpI基因在昆虫细胞中获得表达,表达产物在培养72h达到高峰,重组蛋白的相对分子质量约为58000和70000,与理论值相符,蛋白质加工与天然蛋白类似。动物实验结果表明,重组蛋白具有较好的免疫原性,可刺激小鼠产生中和抗体。SDS—PAGE检测纯化的重组蛋白,纯度达80％。纯化蛋白经western-blot和ELISA检测后显示,具有特异的抗体结合活性。结论 应用昆虫细胞表达水痘-带状疱疹病毒gpI基因,可为水痘-带状疱疹病毒抗原定量分析、糖蛋白ELISA的研制和制备亚单位疫苗提供基础。     

蛋白质组研究技术平台的建立——双向电泳条件的建立及优化

目的　建立和优化蛋白质组研究的双向电泳及相关技术。方法　以固相pH梯度等电聚焦为第一向和垂直SDS PAGE为第二向 ,对样品制备、上样量、IPG胶条、等电聚焦和聚丙烯酰胺凝胶电泳程序及参数、染色方法等相关技术进行了比较研究和条件优化。结果及结论　成功地得到了人胚肺成纤维细胞和蚕卵蛋白质组双向电泳图谱 ,建立了蛋白质组研究的双向电泳技术平台。     

刀额新对虾变应原的分离、鉴定与纯化

目的通过对我国常见的刀额新对虾变应原蛋白进行分离、鉴定与纯化，以提供虾特异性变应原用于食物变态反应疾病的诊断和治疗。方法通过十二烷基硫酸钠一聚丙烯酰胺凝胶电泳（SDS—PAGE）分离刀额新对虾的蛋白质组分并测定其分子量，收集过敏病人血清，采用免疫印迹（Western—blotting）法鉴定其变应原成分，通过离子交换层析对刀额新对虾变应原进行初步纯化，通过疏水层析和凝胶过滤层析进一步对变应原纯化。结果刀额新对虾有17条蛋白带，其中主要条带有10条，68和36kD为可与虾过敏性病人血清IgE结合的特异性变应原；通过各种层析方法纯化出刀额新对虾68kD变应原，纯度为96％。结论对刀额新对虾变应原进行分离、鉴定和纯化，得到高纯度的68kD刀额新对虾变应原。为临床虾变态反应疾病的诊断和治疗奠定基础。     

重组人内皮抑素的表达纯化及对内皮细胞增殖的影响

目的探讨融合表达的重组人内皮抑素对人脐带静脉血管内皮细胞（ECV304）增殖的影响。方法将pTRX—endo质粒转化大肠杆菌BL21（DE3）,IPTG诱导表达,经纯化后加入ECV304细胞培养,MTT法检测对内皮细胞增殖的影响。结果经SDS—PAGE分析,出现特异性蛋白质条带,纯化后得到纯度在95％以上的重组人内皮抑素蛋白,明显抑制ECV304的增殖。结论内皮抑素可明显抑制内皮细胞增殖,存在剂量效应关系,本实验为探索内皮抑素抑制血管生成的机制及应用研究打下基础。     

白纹伊蚊3日龄成蚊饱血前后唾液腺总蛋白谱

目的探讨3日龄白纹伊蚊雌蚊吸食糖水组和饱血组唾液腺总蛋白谱的差异。方法分别采用Pierce蛋白定量试剂盒和SDS-PAGE电泳分析白纹伊蚊3日龄吸食糖水组和饱血组成蚊唾液腺蛋白质浓度和蛋白谱。结果糖水组蚊虫单对唾液腺蛋白质质量约为（1．243Э0．460）μg,饱血组约为（0．945±0．460）μg;SDS—PAGE检测到糖水组唾液腺总蛋白有12条主带,糖水组与饱血组唾液腺总蛋白条带没有变化。结论白纹伊蚊雌蚊饱血后唾液腺总蛋白无特异条带诱导,但饱血后单对唾液腺总蛋白下降。     

Effect of proteins from beef, pork, and turkey meat on plasma and liver lipids of rats compared with casein and soy protein

OBJECTIVE: We assessed the effect of dietary proteins isolated from beef, pork, and turkey meat on concentrations of cholesterol and triacylglycerols in plasma, lipoproteins, and liver and the composition of the microsomal membrane (fatty acids, phosphatidylcholine/phosphatidylethanolamine ratio) compared with that of casein and soy protein in rats. METHODS: Five groups of 12 rats each were fed semisynthetic diets for 20 d that contained 200 g/kg of proteins isolated from beef, pork, or turkey meat or, as controls, casein or soy protein. RESULTS: Rats fed beef, pork, or turkey proteins did not differ in cholesterol concentrations of plasma, lipoproteins, and liver and in composition of microsomal membrane from rats fed the casein diet. All groups fed a protein from an animal source had higher very low-density lipoprotein (VLDL) and liver cholesterol concentrations than did rats fed soy protein. However, rats fed pork protein had lower concentrations of triacylglycerols in liver, plasma, and VLDL and lower mRNA concentrations of sterol regulatory element binding protein-1 and glucose-6-phosphate dehydrogenase than did rats fed casein. However, concentrations of plasma and VLDL triacylglycerols in rats fed pork protein were not as low as those observed in rats fed soy protein. CONCLUSION: Proteins isolated from beef, pork, or turkey meat do not differ from casein in their effects on cholesterol metabolism. Pork protein decreases plasma triacylglycerol concentrations compared with casein but not compared with soy protein. The triacylglycerol-lowering effect of pork protein compared with casein is suggested to be caused by decreased hepatic fatty acid synthesis.     

Fresh and fresh lean pork are substantial sources of key nutrients when these products are consumed by adults in the United States

Many fresh pork products, in particular, fresh lean pork products, are nutrient-dense sources of protein and several other nutrients. The purpose of this study was to estimate nutritional contributions of fresh and fresh lean pork to adults' diets in the United States. Mean total nutrient intakes by fresh and fresh lean pork consumers on a day of recall were compared with intakes by nonconsumers to test the hypothesis that overall nutrient intakes by consumers were comparable with or better as compared with intakes by nonconsumers. Intakes were assessed using the National Health and Nutrition Examination Survey 2003 to 2006. Based on 1 day of dietary intake, 10% of adults consumed fresh pork, and 4% consumed fresh lean pork. Among consumers, fresh and fresh lean pork contributed 16% and 9%, respectively, of total fat and accounted for 23% to 31% of total protein, cholesterol, selenium, and thiamin intake. Fresh and fresh lean pork also accounted for 11% to 19% of total saturated fat, phosphorus, potassium, riboflavin, niacin, vitamin B₆, and vitamin B₁₂ in the diets of consumers and contributed 21% and 16%, respectively, of total zinc. Diets including fresh or fresh lean pork provided higher energy-adjusted amounts of protein, selenium, thiamin, and vitamin B₆ as compared with diets of adults not consuming fresh pork (P < .05) and provided comparable amounts of fat and saturated fat. Consumption of lean cuts of fresh pork is consistent with dietary guidance, and selection of fresh lean pork products by current nonconsumers could increase dietary variety without adversely affecting nutrient intake.     

Processed pork is the most frequently consumed type of pork in a survey of Australian children

Pork represents a core food that provides key nutrients to the diet. Dietary guidelines recommend limiting processed meat intake because of adverse health outcomes. The aims of this study were to describe pork consumption, assess the contribution of pork to nutrient intakes, and compare anthropometric characteristics between pork consumers and nonconsumers in a survey of Australian children. We hypothesized that pork consumption will contribute to intakes of key nutrients and that the weight status of children who consume pork will be similar to nonconsumers. This study involved a secondary analysis of the 2007 Australian National Children's Nutrition and Physical Activity Survey. Pork and pork-containing dishes were identified and classified as fresh or processed pork. The contributions of pork to nutrient intakes were calculated. Weight, waist circumference, and body mass index were compared between pork consumers and nonconsumers. Data from 4487 children were available for use. Of this sample, 2245 reported consuming pork, 14% (n = 310) of whom consumed fresh pork, whereas 93% (n = 2084) consumed processed pork. All types of pork contributed to intakes of protein, niacin, and zinc. In addition, fresh pork contributed to intakes of thiamine, long-chain omega-3, phosphorous, and potassium. Total and processed pork contributed 12.2% and 13.0% of sodium, respectively. There were no significant differences between weight, waist circumference, and body mass index in consumers and nonconsumers of total, fresh, or processed pork. In a survey of Australian children, processed pork was the most frequently consumed form of pork, suggesting a deviation from dietary guidelines.     

Effects of eating fresh lean pork on cardiometabolic health parameters

High protein meat-based diets are commonly promoted for weight loss, supposedly by increasing satiety and energy expenditure. Pork is a good source of protein however little information on the metabolic effects of pork consumption exists. This pilot study aimed to examine whether regular consumption of fresh lean pork could improve body composition and cardiovascular risk factors in a 6 month parallel intervention trial. 164 overweight adults (mean BMI 32) were randomly assigned to incorporate up to 1 kg pork/week by substituting for other foods or maintain their habitual diet (control). Plasma levels of lipids, glucose and insulin, BMI, waist/hip circumference, blood pressure, heart rate and arterial compliance were measured at baseline and 3 and 6 months. Body composition was determined using dual energy X-ray absorptiometry. A total of 144 volunteers completed and volunteers in the pork group increased their intake 10 fold by substituting pork for mainly beef and chicken. After 3 months, there were significant (p ≤ 0.01) reductions in weight, BMI, waist circumference, % body fat, fat mass and abdominal fat in the pork group relative to controls, which persisted for 6 months. There was no change in lean mass, indicating that the reduction in weight was due to loss of fat mass. There were no significant effects on other metabolic parameters. Regular consumption of lean fresh pork may improve body composition.     

Effect of dietary vegetable and animal proteins on atherothrombosis in mice

OBJECTIVE: It is believed that vegetable and fish, but not animal, proteins prevent thrombosis. The present study compared the effect of long-term intake of purified vegetable and animal proteins (casein, pork, egg white, chicken, white and red fish, soybean, and potato) and powders from whole vegetable and animal meats (soybean, pork, chicken, and horse mackerel) on thrombotic tendency. METHODS: Western-style high-fat diets with various proteins were given to mice deficient in apolipoprotein-E and low-density lipoprotein receptor for 8 or 12 wk. Thrombotic tendency was assessed by a laser-induced thrombosis test, severity of atherosclerosis was assessed by morphometric analysis of the aortic lesions, and reactivity of platelets was measured by a shear-induced platelet function test. RESULTS: There was no difference in thrombotic tendency between the vegetable and animal protein diets with the exception of pork protein, which showed a prothrombotic effect. The latter was not observed in animals kept on a high-protein diet. Thrombotic tendency was not affected by preparation of dietary powders from whole pork, chicken, or horse mackerel meats and casein (control), all of which contained the same percentages of protein and lipid. In contrast, soybean powder showed an antithrombotic effect, which was mainly due to an inhibition of atherogenesis rather than to platelet inhibition. CONCLUSION: Neither the purified animal nor the vegetable protein diet affected thrombotic tendency. Soybean powder inhibited atherothrombosis mainly by inhibiting the atherosclerotic process.     

Pork, beef and chicken have similar effects on acute satiety and hormonal markers of appetite

The effects of three different meat-containing breakfast meals (pork, beef or chicken) on acute satiety and appetite regulatory hormones were compared using a within-subjects study design. Thirty fasting non-smoking pre-menopausal women attended a research centre on three test days to consume, a meat-containing meal matched in energy (kJ) and protein content, palatability, and appearance. No difference was found between meat groups for either energy intake or macronutrient profile of food consumed at a subsequent ad libitum buffet lunch, or over the rest of the day. Visual Analogue Scale (VAS) ratings for hunger and satiety over an 180 min period did not differ between test meals. After consumption of the test meals, a significant difference was found in PYY response between pork and chicken meals (P=0.027) but not for levels of CCK, ghrelin, insulin or glucose. This study positions pork, beef, and chicken as equal in their effect on satiety and release of appetite-related intestinal hormones and of insulin.     

Effects of Dietary Protein Source and Quantity during Weight Loss on Appetite,Energy Expenditure,and Cardio-Metabolic Responses

Higher protein meals increase satiety and the thermic effect of feeding (TEF) in acute settings, but it is unclear whether these effects remain after a person becomes acclimated to energy restriction or a given protein intake. This study assessed the effects of predominant protein source (omnivorous, beef/pork vs. lacto-ovo vegetarian, soy/legume) and quantity (10%, 20%, or 30% of energy from protein) on appetite, energy expenditure, and cardio-metabolic indices during energy restriction (ER) in overweight and obese adults. Subjects were randomly assigned to one protein source and then consumed diets with different quantities of protein (4 weeks each) in a randomized crossover manner. Perceived appetite ratings (free-living and in-lab), TEF, and fasting cardio-metabolic indices were assessed at the end of each 4-week period. Protein source and quantity did not affect TEF, hunger, or desire to eat, other than a modestly higher daily composite fullness rating with 30% vs. 10% protein diet (p = 0.03). While the 20% and 30% protein diets reduced cholesterol, triacylglycerol, and APO-B vs. 10% protein (p < 0.05), protein source did not affect cardio-metabolic indices. In conclusion, diets varying in protein quantity with either beef/pork or soy/legume as the predominant source have minimal effects on appetite control, energy expenditure and cardio-metabolic risk factors during ER-induced weight loss.     

Consumption and nutritional quality of grilled pork purchased from open road-side restaurants of Benin

This study focused on the determination of consumption habits and nutritional composition of ready-to-eat grilled pork from Benin. A face-to-face survey of 372 grilled pork consumers using a semi-structured questionnaire was carried out in the same municipalities where 24 samples of grilled pork were collected for physico-chemical characterisation. Three hundred out of 372 (80.6 %) consumers eating grilled pork at least once per month were selected to estimate the daily intake of protein, lipid and fatty acids. Grilled pork consumption ranged between 0.7 g/day and 342.9 g/day with a median of 28.6 g/day. Protein and lipid contents ranged between 19.5 % and 69.5 % of dry weight and between 27.9 % and 82.1 % of dry weight, respectively. Fatty acid profile showed that linoleic acid was the most abundant omega 6-PUFA (Polyunsaturated fatty acid). The consumption of high quantity of grilled pork (342.9 g/day) enables to cover 25 % (for women) and 31 % (for men) of adult requirement of PUFA (11−22 g/day for women and 14−28 g/day for men). The omega 6 daily intake from high grilled pork consumption contributed to satisfy about 15 % of adult requirement of omega 6 fatty acids for both genders while the omega 3 daily intake from high grilled pork consumption was over 80 % for both genders.     

Pork and Chicken Meals Similarly Impact on Cognitive Function and Strength in Community-Living Older Adults: A Pilot Study

A pilot quasi-experimental study investigated whether provision of pork, a rich source of thiamin, as the main protein source in meals four times/week for 12 weeks resulted in improved muscle mass, body strength, and cognitive function in community-living older adults compared to similar meals containing chicken. Retirement villages were randomized to receive pre-prepared frozen meals containing either pork or chicken. Dietary intake was assessed by three-day food records and cognitive domains assessed using validated tests. Hand grip strength was measured and lower extremity performance assessed by the sit-to-stand test, get-up-and-go test and six-minute walk test. Forty-eight volunteers participated (78.2 ± 6.2 y). In linear mixed models, controlling for baseline physical activity and dietary protein and energy intake, no differences were found between pork (n = 19) and chicken (n = 12) groups. The chicken group had improved Rey Auditory Verbal Learning test scores (verbal learning and memory) at six weeks (p < 0.001). Provision of four pork meals a week did not result in improvements in cognitive function, nor measures of strength or physical function, compared to those receiving chicken meals in healthy older adults. This suggests that merely changing the type of dietary protein provided by meat does not impact physical or cognitive function.     

Amino Acid Availability in Man

Beef protein scores highest in terms of nitrogen and amino acid availability when compared to milk and egg, cottage cheese, pork, milk alone, peanut butter and the control whole-egg protein.