猪生殖和呼吸综合征中国分离株ORF7基因的克隆及鉴定

本研究利用对应于ＡＴＣＣＶＲ－２３３２及ＬＶＯＲＦ７基因保守序列的１对均为２８个碱基的引物１００１１ＰＣＳ、１０１０ＰＣＲ对ＰＲＲＳＶ中国分离株Ｂ１３进行ＲＴ－ＰＣＲ，结果扩增出了１条包括完整ＯＲＦ７基因的５１０ｂｐ的ＤＮＡ片段。纯化此扩增产物，并对其进行ＥｃｏＲＩ／ＰｓｔＩ双酶切，与用ＥｃｏＲＩ、ＰｓｔＩ双酶切及碱性磷酸酶处理的ＰＵＣ１８载体连接。转化大肠杆菌。结果得到了１个Ｂ１３ＯＲＦ７基因与ＰＵＣ１８载体的重组质粒ＰＵＣ１８Ｂ１３ＯＲＦ７１０。通过ＥｃｏＲＩ／ＰｓｔＩ及ＰＣＲ证明此重组质粒即为Ｂ１３ＯＲＦ７基因与ＰＵＣ１８载体的重组质粒。从而为ＯＲＦ７基因及所表达的核衣壳蛋白进一步研究奠定了基础。     

IBDV VP2/VP243基因DNA疫苗表达质粒的构建与表达

将传染性法氏囊病病毒（ＩＢＤＶ）ＶＰ２／ＶＰ２４３插入真核表达载体ｐｃＤＮＡ中,置于ＣＭＶ启动子的调控之下,构建成ＤＮＡ疫苗表达质粒ｐｃＤＮＡ ＶＰ２和ｐｃＤＮＡ ＶＰ０。分别转染ＣＥＦ细胞后,于２４、４８、７２ｈ取细胞裂解液进行ＥＬＩＳＡ、ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎ ｂｌｏｔ检测,ｐｃＤＮＡ ＶＰ２于转染后２４ｈ呈阳性反应,４８ｈ表达量高于２４ｈ;ｐｃＤＮＡ ＶＰ０于转染后４８ｈ呈阳性反应     

组织型纤溶酶原激活剂cDNA在BHK21细胞中的表达

将组织型纤溶酶原激活剂（ｔ－ＰＡ）基因ｃＤＮＡ经多次亚克隆插入到真核表达载体ｐＳＶＬ的ＳＶ４０启动子和ｐｃＤＮＡ３的ＣＭＶ启动子下游,构建了重组表达质粒ｐＳＶＬ－ｔＰＡ和ｐｃＤＮＡ３－ｔＰＡ。分别将这２种表达质粒以脂质体载体法转染ＢＨＫ２１细胞,ＥＬＩＳＡ检测结果表明,ｐＳＶＬ－ｔＰＡ和ｐｃＤＮＡ３－ｔＰＡ在哺乳动物细胞中能够表达,７２ｈ表达量分别是５２．８μｇ／Ｌ和７０．４μｇ／Ｌ。     

猪生殖与呼吸综合征病毒ORF6片段的cDNA克隆及鉴定

根据已发表的ＰＲＲＳＶ基因组序列，设计并合成了１对特异扩增ＡＴＣＣ ＶＲ－２３３２株的ＯＲＦ６的基因的引物，以ＡＴＣＣ ＶＲ－２３３２基因组为模板，通过ＲＴ－ＰＣＲ扩增出５８６ｂｐ的ｃＤＮＡ产物。将该ｃＤＮＡ片段经Ｔ－Ａ连接插入ｐＧＥＭ－Ｔｅａｓｙ载体中，用重组质粒转化大肠埃希氏菌ＪＭ１０９，获得重组质粒转化菌。对重南粒ＤＮＡ进行ＰＣＲ及酶切鉴定，证明插入的ＤＮＡ片段与ＰＣＲ扩增的ＤＮＡ片段大小相     

pCMV—TNFα的构建及其在动物细胞中的表达

以Ｘｂａ Ｉ和Ｅｃｏ ＲＶ双酶消化ｐＢＴ１获得肿瘤坏死因子α（ＴＮＦα）ｃＤＮＡ基因片段，以Ｘｂａ Ｉ和Ｓｍａ Ｉ酶切点定向克隆入质粒载体ｐＣＭＶ４，构建了人ＴＮＦα重组质粒ｐＣＭＶ－ＴＮＦα。采用ＤＮＡ－磷酸钙共沉淀法转染ＣＯＳ７和ＮＩＨ３Ｔ３细胞，收集转染后不同时间的细胞培养上清，检测ＴＮＦα的表达水平和生物学活性。ＥＬＩＳＡ检测表明，两种细胞转染后，其培养上清中均有ＴＮＦα抗原存在，而载体等     

NDV融合蛋白裂解位点基因的重组昆虫杆状病毒表达载体的构建…

将新城疫病毒（ＮＤＶ）Ｆ４６Ｅ９株和ＬａＳｏｔａＥ４株的融合蛋白裂解位点（Ｆｃ）基因从本实验室构建的重组质粒ｐＵＣＦ４６Ｆｃ和ｐＵＣＬａＦｃ中用ＥｃｏＲＩ和ＳａｌＩ切出。将这２个毒株的Ｆｃ基因定向克隆入昆杆状病毒表达载体ｐＳＸＩＶＶＩＸ３的ＥｃｏＲＩ和ＳａｌＩ位点之间，获得２个毒株的Ｆｃ基因克隆ｐＸ３Ｆ４６Ｆｃ和ｐＸ３ＬａＦｃ。重组质粒用ＥｃｏＲＩ／ＳａｌＩ，ＰｓｔＩ酶切法，ＰＣＲ和核酸探针法进行     

猪瘟病毒囊膜糖蛋白DNA疫苗的研制

构建了含有猪瘟病毒石门株囊膜糖蛋白（Ｅ２）基因的重组真核表达质粒ｐＲＣＳＥ２。将ｐＲＣＳＥ２和空载体ｐＲＣ分别免疫小鼠,ＥＬＩＳＡ检测结果表明：仅ｐＲＣＳＥ２免疫鼠可以产生猪瘟病毒的特异抗体。ＤＮＡ疫苗在许多传染病研究中已取得可喜成果,因此猪瘟病毒ＤＮＡ疫苗可能会成为防制猪瘟的一条新的途径。     

NDV融合蛋白裂解位点基因的重组昆虫杆状病毒表达载体的构建与鉴定

将新城疫病毒（ＮＤＶ）Ｆ４６Ｅ９株和ＬａＳｏｔａＥ４株的融合蛋白裂解位点（Ｆｃ）基因从本实验室构建的重组质粒ｐＵＣＦ４６Ｆｃ和ｐＵＣＬａＦｃ中用ＥｃｏＲＩ和ＳａｌⅠ切出。将这２个毒株的Ｆｃ基因定向克隆入昆虫杆状病毒表达载体ｐＳＸＩＶＶＩ＋Ｘ３的ＥｃｏＲＩ和ＳａｌⅠ位点之间，获得２个毒株的Ｆｃ基因克隆ｐＸ３Ｆ４６Ｆｃ和ｐＸ３ＬａＦｃ。重组质粒用ＥｃｏＲＩ／ＳａｌⅠ、ＰｓｔⅠ酶切法、ＰＣＲ和核酸探针法进行了鉴定，证明插入的基因来自ｐＵＣＦ４６Ｆｃ和ｐＵ－ＣＬａＦｃ，插入的位置和方向都正确。这２个载体的构建为Ｆｃ基因在昆虫细胞系统的表达创造了条件。     

猪生殖-呼吸道综合征病毒CH-1_株N基因的原核表达

将ＰＲＲＳＶＣＨ１ａ株Ｎ基因用ＥｃｏＲＩ和ＰｓｔＩ双酶切从重组质粒ｐＵＣ１８ＯＲＦ７切下后，插入到原核表达载体ｐＢＶ２２０的ＰＲ、ＰＬ启动子下游，得到重组表达质粒ｐＢＶ２２０ＯＲＦ７。转化了ｐＢＶ２２０ＯＲＦ７的大肠杆菌ＪＭ８３经诱导培养后，用ＳＤＳＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔ检测表达产物。结果表明ＰＲＲＳＶＣＨ１ａ株的Ｎ基因在原核载体上得到高效表达，表达产物占菌体总蛋白的１５３％。表达蛋白可望成为有价值的ＰＲＲＳ诊断抗原。     

鸡传染性喉气管炎病毒王岗株gC和gD基因的克隆及序列分析

根据已发表的传染性喉气管炎病毒（ＩＬＴＶ）ＳＡ－２株的核酸序列,设计了１对引物,以ＩＬＴＶ－中国王岗株ＤＮＡ为模板,ＰＣＲ法扩增出１条１．３９ｋｂ的基因片段,将扩增产物插入到真核表达质粒ｐＣＲ＾ＴＭ３－Ｕｎｉ,得到重组质粒ｐＴＡ－ｇＤ,另将克隆到ｐＢｌｕｅｓｃｒｉｐｔ ＳＫ质料中的ｇＣ基因酶毁后,插入到ｐＣＲ＾ＴＭ３－Ｕｎｉ载体,得到重组质粒ｐＴＡ－ｇＣ,经电泳分析、酶分、ＰＣＲ鉴定后,进行序列测     

Effect of nutrient restriction on calpain and calpastatin content of skeletal muscle from cows and fetuses

Calpains are crucial for the degradation of myofibrillar proteins in muscle. Calpastatin is a specific inhibitor of calpains. The objective of this study was to elucidate the effect of nutrient restriction on the activity of calpains and calpastatin in the skeletal muscle of both cows and fetuses. Beginning 30 d after conception, 20 cows were fed either a control diet consisting of native grass hay fortified with vitamins and minerals at recommendations for a mature cow to gain 0.72 kg/d or half the vitamins and minerals and millet straw at 68.1% of NEm requirements. Cows were slaughtered on d 125 of gestation, and the LM was sampled at the 12th rib for calpain and calpastatin measurement. When comparing the muscle samples from nutrient-restricted and control cows, no difference in the activity of calpain I and II was observed; however, there was a significant difference (P < 0.05) in calpastatin activity. Muscle samples from control cows had greater calpastatin content than those of nutrient-restricted cows (P < 0.05); in contrast, the calpastatin content of fetal muscle was greater in fetuses gestated by nutrient-restricted cows than those of control cows (P < 0.05). Further, there were three calpastatin isoforms of 125, 110, and 70 kD detected in fetal muscle, whereas only the110-kD isoform was detected for cow muscle. These results indicate that the activity of the calpain system in skeletal muscle is mainly controlled through the expression of calpastatin. Alternating the calpastatin content in muscle and thereby modulating calpain activity may provide a mechanism for the maintenance of fetal muscle growth during nutrient restriction, whereas skeletal muscle loss in cows is upregulated.     

钙蛋白酶系统对猪肉嫩度影响的研究进展

嫩度是影响猪肉品质的重要性状之一,肉品的嫩度取决于肌肉结构及其相关蛋白质的变化程度。影响肌肉嫩度的因素有很多,其中钙蛋白酶系统在肌肉嫩化过程中发挥着重要作用。钙蛋白酶抑制蛋白是钙蛋白酶系统的成员之一,同时也为钙蛋白酶抑制蛋白基因的表达产物,钙蛋白酶抑制蛋白基因为调控猪肉品质的候选基因之一。作者对影响肌肉嫩度的因素、钙蛋白酶系统的结构和生理功能,以及钙蛋白酶系统和钙蛋白酶抑制蛋白基因型对猪肉嫩度的影响进行综述。     

Intermuscular variation in tenderness: association with the ubiquitous and muscle-specific calpains

The biochemistry of intermuscular variation in tenderness is not fully understood. To investigate the role of the calpains in this process we performed two experiments using bovine and ovine species. In the bovine experiment, two distinct muscles, longissimus thoracis et lumborum (LT) and psoas major (PM), were used. In the ovine experiment, four muscles, LT, PM, semimembranosus (SM), and semitendinosus (ST), were used. Muscles were sampled at death for the determination of the steady-state mRNA level of calpains and calpastatin and the activities of calpain 1, 2, and calpastatin. Muscles were also sampled to determine the temporal changes in pH, tenderness, and the activity of the ubiquitous calpain system during postmortem aging. The results of the relative rate of tenderization in both species was found to be related to muscle type; LT had the highest value in both species. Within species, the mRNA steady-state levels of calpain 1 and calpastatin were similar in various bovine and ovine muscles. Bovine calpain 2 mRNA level was significantly lower in the LT than in the PM. Ovine calpain 2 mRNA level was lower, but not significantly different, in the LT compared to the other muscles. The mRNA level of bovine calpain 3 was significantly higher in the LT muscle than in the PM. In the ovine, the mRNA level of calpain 3 was highest in the LT, followed by SM, PM, and ST. Results on the activity of the ubiquitous calpain system in various muscles at death were dependent on muscle type and species. Temporal changes in the activity of calpains and calpastatin during the first 24 h of postmortem aging were similar in the muscles studied: calpain 1 and calpastatin declined significantly and calpain 2 remained relatively unchanged. The temporal changes in muscle pH in both experiments indicated that the extent and rate of pH decline during aging was related to muscle type. Correlation analysis between the relative rate of tenderization and mRNA expression of calpains revealed a strong relationship with calpain 3 in both species.     

钙蛋白酶系统研究进展

钙蛋白酶系统主要是由钙蛋白酶类和钙蛋白酶抑制蛋白组成。钙蛋白酶细胞内主要的蛋白水解酶,参与神经发育、肌纤维降解、信号传导等过程。因此,钙蛋白酶系统在肌肉生长、改善肉的嫩度及治疗某些疾病等方面起着重要的作用。本文主要就其成员、结构、功能等方面作一综述。     

Effects of age and castration on activities of calpains and calpastatin in sheep skeletal muscle.

The objectives of this experiment were to assess effects of animal age and castration on activities of calpain I, calpain II, and calpastatin in sheep skeletal muscle. Ten newborn male lambs (2.9 kg), six weaned wethers (23.2 kg), six weaned rams (22.2 kg), six market wethers (55.4 kg), and six market rams (60.2 kg) were slaughtered and samples of biceps femoris were taken for assay of calpain I (micromolar calcium-dependent proteinase), calpain II (millimolar calcium-dependent proteinase), and calpastatin. Preweaning weight gain was similar for rams and wethers; however, postweaning ram growth exceeded (P less than .05) that of wethers. Ram biceps femoris weights at market were greater than those of wethers (P less than .05). Irrespective of age or gender, activity of calpain II was two- to threefold greater than that of calpain I. Muscle calpastatin activity was severa fold higher than calpain I and II activities. Activities of calpains and calpastatin declined (P less than .05) between birth and weaning. A portion of these losses were due to a dilution effect caused by accumulation of muscle proteins. Neonatal attenuation of calpain activities may underlie age-related attenuation of fractional rates of muscle protein degradation. Although ram muscle growth exceeded that of wethers, no differences (P greater than .05) in activities of muscle calpains or calpastatin were detected between these two groups at weaning or at market weight. Hence, castration did not influence lamb muscle growth by altering muscle calpain or calpastatin activities.     

Quantification of ovine and bovine calpain I, calpain II, and calpastatin mRNA by ribonuclease protection assay.

We developed a ribonuclease protection assay (RPA) to quantify the mRNA mass of calpastatin and the catalytic subunits of calpains I and II in ovine and bovine tissues. The method is based on constructing standard curves using predetermined amounts of in vitro synthesized sense cRNA of the calpain system, hybridized with excess radiolabeled antisense counterprobes. This is possible because the vectors used for riboprobe preparation can be used to transcribe the sense cRNA required to generate the standard curves to quantify absolute calpain I, calpain II, and calpastatin mRNA levels. We used the RPA to study calpain I, calpain II, and calpastatin gene expression in ovine liver, heart, and skeletal muscle. The results revealed that calpain II gene expression was similar in the three tissues. However, the expression of calpain I and calpastatin genes indicates that each tissue has its unique pattern. We also analyzed the activity of calpain I, calpain II, and calpastatin by the conventional DEAE chromatographic method for comparison. The results indicated that the RPA is more repeatable than the DEAE method. Special features of the RPA as compared with DEAE chromatography are as follows; 1) the RPA is a reliable method for quantifying the expression of calpains in all tissues because it is not affected by the presence of inhibitors or activators, 2) the RPA method can be expanded to analyze the expression of the tissue-specific calpains simply by designing specific probes for them, and 3) the RPA requires a small amount of tissue. The described method will facilitate future studies on the gene expression of calpains and will contribute to determining their physiological functions.     

Relationship between beta-adrenergic agonists,calpain system activity and beef texture: A systematic review

The objective was to conduct a systematic review to evaluate the effects of dietary supplementation with beta-adrenergic agonists on calpains and calpastatin activity in bovine muscle and changes in meat tenderness. A survey was conducted in June 2019 on Science Direct, Web of Science, Scopus, PubMed and Capes Periodicals, using four keyword combinations: agonist and calpain and cattle; agonist and calpain and bovine; agonist and calpain and heifers; agonist and calpain and steers. Thirteen studies were selected, 54% concluded that supplementation with beta-adrenergic agonists increases calpastatin activity, 23% observed increase in their gene expression and 23% reported no effect on activity or expression of this enzyme. Nine studies evaluated the influence of beta-adrenergic agonists supplementation on meat texture and all found an increase in shear force values. There is strong evidence that beta-adrenergic agonists may increase calpastatin activity in the muscle, causing damage to meat tenderness.     

Isolation and characterization of mu-calpain, m-calpain, and calpastatin from postmortem muscle. I. Initial steps

Evidence has indicated that mu-calpain, m-calpain, and calpastatin have important roles in the proteolytic degradation that results in postmortem tenderization. Simple assays of these 3 proteins at different times postmortem, however, has shown that calpastatin and mu-calpain both rapidly lose their activity during postmortem storage, so that proteolytic activity of mu-calpain is nearly zero after 3 d postmortem, even when assayed at pH 7.5 and 25 degrees C, and ability of calpastatin to inhibit the calpains is 30% or less of its ability when assayed at death. m-Calpain, however, retains much of its proteolytic activity during postmortem storage, but the Ca(2+) requirement of m-calpain is much higher than that reported to exist in postmortem muscle. Consequently, it is unclear how the calpain system functions in postmortem muscle. To clarify this issue, we have initiated attempts to purify the 2 calpains and calpastatin from bovine semitendinosus muscle after 11-13 d postmortem. The known properties of the calpains and calpastatin in postmortem muscle have important effects on approaches that can be used to purify them. A hexyl-TSK hydrophobic interaction column is a critical first step in separating calpastatin from the 2 calpains in postmortem muscle. Dot-blot assays were used to detect proteolytically inactive mu-calpain. After 2 column chromatographic steps, 5 fractions can be identified: 1) calpastatin I that does not bind to an anion-exchange matrix, that does not completely inhibit the calpains, and that consists of small polypeptides <60 kDa; 2) calpastatin II that binds weakly to an anion-exchange matrix and that contains polypeptides <60 kDa; all these polypeptides are smaller than the native 115- to 125-kDa skeletal muscle calpastatin; 3) proteolytically active mu-calpain even though very little mu-calpain activity can be detected in zymogram assays of muscle extracts from 11- to 13-d postmortem muscle; this mu-calpain has an autolyzed 76-kDa large subunit but the small subunit consists of 24-, 26- and a small amount of unautolyzed 28-kDa polypeptides; 4) proteolytically active m-calpain that is not autolyzed; and 5) proteolytically inactive mu-calpain whose large subunit is autolyzed to a 76-kDa polypeptide and whose small subunit contains polypeptides similar to the proteolytically active mu-calpain. Hence, loss of calpastatin activity in postmortem muscle is due to its degradation, but the cause of the loss of mu-calpain activity remains unknown.     

Effect of pH and ionic strength on mu- and m-calpain inhibition by calpastatin

The objectives of this study were to determine the extent to which pH and ionic strength influence mu- and m-calpain activity and the inhibition of calpains by calpastatin. Calpastatin, mu-calpain, and m-calpain were purified from at-death porcine semimembranosus. Mu-calpain or m-calpain (0.45 U) were incubated with the calpain substrate Suc-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin in the presence of calpastatin (0, 0.15, or 0.30 U of calpain inhibitory activity) under the following pH and ionic strength conditions: pH 7.5 and 165 mM NaCl or 295 mM NaCl; pH 6.5 and 165 mM NaCl or 295 mM NaCl; and pH 6.0 and 165 mM NaCl or 295 mM NaCl. The reactions were initiated with addition of 100 microM (mu-calpain) or 1 mM CaCl2 (m-calpain), and calpain activity was recorded at 30 and 60 min. Mu-calpain had the greatest (P < 0.01) activity at pH 6.5 at each ionic strength. Higher ionic strength decreased mu-calpain activity (P < 0.01) at all pH conditions. Inhibition percent of mu-calpain by calpastatin was not affected by pH; however, it was influenced by ionic strength. Inhibition of mu-calpain by calpastatin was higher (P < 0.01) at 295 mM NaCl than at 165 mM NaCl when 0.3 units of calpastatin were included in the assay. Activity of m-calpain was greater (P < 0.01) at pH 7.5 than at pH 6.5. m-Calpain activity was not detected at pH 6.0. Inhibition of m-calpain was greater (P < 0.01) when 0.15 and 0.3 U calpastatin were added at pH 6.5 than 7.5 at 165 mM NaCl, whereas percentage inhibition of m-calpain was greater (P < 0.01) at 295 mM than 165 mM NaCl at pH 7.5 and 6.5. These observations provide new evidence that defines further the influence of pH decline and increased ionic strength on mu-calpain, m-calpain, and calpastatin activity, thereby helping to more accurately define a role for these enzymes in the process of postmortem tenderization.     

钙蛋白酶系统在肌肉生长和肉品嫩化方面的研究

钙蛋白酶系统主要由钙蛋白酶(calpain)及钙蛋白酶抑制蛋白(calpastatin)组成,calpain是存在于细胞质中的依赖于Ca2+的中性蛋白酶,calpastatin是钙蛋白酶的内源抑制蛋白。近年的研究表明,calpain是细胞质中主要的蛋白水解酶,在肌原纤维蛋白降解中起着重要的作用。肌肉增长和宰后嫩度的变化与蛋白质水解程度密切相关。因此,钙蛋白酶系统的活性会影响畜禽肌肉增长和肉的嫩度。文中综述了钙蛋白酶系统各种酶的结构及其如何在肌肉生长和肉的嫩化中起作用。