转基因猪在武汉问世

6月27日,湖北省农科院对外公布:含人类基因、其血液能取代人血提取"医用白蛋白”的"转基因猪”正式问世武汉. 湖北省农科院生物所转基因课题组经过10多年的研究,成功地在国内首次用转基因猪表达人的血清蛋白.打日,在该所的试验猪场里,记者见到3头转基因猪.华中农业大学生命科学院测定,其中蛋白最高表达量为20.3g/L,是目前国内外转基因动物肝脏表达外源蛋白的最高水平.     

hApoCⅢ转基因小型猪的脂蛋白构成变化

为分析hApoCⅢ转基因小型猪的脂蛋白构成变化,探讨载脂蛋白ApoCⅢ在代谢调节中的作用,应用Western blot和免疫组织化学方法检测人ApoCⅢ在转基因小型猪体内的表达和分布,并通过脂蛋白的快速蛋白液相色谱分析试验分析转基因小型猪的脂蛋白构成特点。结果显示,在转基因小型猪的肝脏和小肠组织中能检测到hApoCⅢ蛋白,且转基因小型猪血浆极低密度脂蛋白(VLDL)颗粒较非转基因小型猪显著增高。这表明,hApoCⅢ转基因小型猪过表达ApoCⅢ蛋白造成其VLDL颗粒显著增高。     

F1代转pGH、IGF-1双基因猪的阳性鉴定及其差异性表达

为了探究转pGH、IGF-1双基因猪F1代阳性率及表达差异情况,试验采用PCR、测序及荧光定量PCR检测转pGH、IGF-1双基因猪的mRNA表达水平。结果表明:53头F1代猪中17头为阳性猪,阳性率为32.08%,证实外源pGH和IGF-1基因已遗传到F1代中;1月龄到4月龄转双基因猪与非转基因猪IGF-1和pGH基因平均表达量均无显著性差异(P0.05);5月龄转双基因猪pGH和IGF-1基因平均表达量分别是非转基因猪的1.98倍和2.00倍,存在显著差异(P0.05);6月龄转双基因猪pGH和IGF-1基因平均表达量分别是非转基因猪的1.67倍和1.73倍(P0.05)。说明pGH、IGF-1双基因已遗传至F1代猪中,并且能够在猪体内稳定表达。     

诱导性表达Cre重组酶转基因猪成纤维细胞的构建及鉴定

通过PCR方法分别从pGL3-Mx1载体上和pcDNA3.1(+)载体上扩增猪Mx1启动子和BGHpolyA;利用重叠PCR方法获得猪源的Cre重组酶基因,并从pGcFRT2NeoR上用Xhol Ⅰ和Sal Ⅰ酶切得到FRT2NeoR盒子.将上述4个片段利用SOE-PCR及T5 DNA连接酶连接,然后利用原核表达载体pET28a(+)构建Cre表达载体Mx1-Cre-BGHpolyA-FRT2neoR.用LipofeetamineTM2000介导转染猪成纤维细胞,G418筛选阳性细胞,PCR鉴定.结果表明,成功构建了诱导性表达Cre重组酶表达载体Mx1-Cre-BGHpolyA-FRT2neoR,并成功整合到猪成纤维细胞的基因组中,获得了诱导性表达Cre重组酶的转基因猪成纤维细胞,为最终通过核移植方法获得诱导性表达Cre重组酶转基因猪奠定基础.     

未来人类供血者——转基因猪

据“养猪信息”晓雨报导:最近美国“DNX”公司已生产出了能表达人类血红蛋白的转基因猪。人类血红蛋白的表达水平占其总血红蛋白的10～15％,如果可以提高表达率,那么转基因猪就可对血液代用品市场     

科技

正国家动保工程中心再获3项转基因生物安全证书2项国家三类新兽药证书2月15日,由国家动保工程中心主持申报的重组大肠杆菌表达鸡α干扰素、重组大肠杆菌表达猪α干扰素、重组大肠杆菌表达鸡传染性法氏囊病VP2蛋白基因工程亚单位疫苗3个项目,历时1年时间,同时获得农业部转基因生物安全证书。2月17日,农业部发布2361号公告,由国家动     

猪腮腺分泌蛋白在腮腺生后发育过程中表达特性研究

腮腺的解剖结构和分泌特点,决定了它具备进行生物反应器和抗病育种研究的良好条件。猪肋腺分泌蛋白(PSP)基因在腮腺中特异性表达,但PSP的表达量尚无报道。本研究利用荧光定量PCR对猪PSP在腮腺生后发育不同阶段的表达进行研究。结果表明:PSP在不同发育期的腮腺中高效表达,而且表达量的变化在可接受的范围内。因此可以利用猪PSP基因的调控区作为转基因生物反应器的转基因平台。     

家畜（禽）转基因研究现状

家畜（禽）转基因研究现状李光鹏严云勤徐立滨（东北农业大学生物工程系·哈尔滨·１５００３０）收稿日期：１９９６－０４－３０１转基因家畜（禽）研究概述１．１猪最早进行的转基因猪研究，是将人生长激素（ｈＧＨ）基因导入猪的基因组中。但ｈＧＨ基因在猪中的表达，...     

猪Myostatin同源重组载体的构建

根据绵羊、猪、小鼠和牛的Myostatin基因序列设计引物,通过PCR方法扩增了猪Myostatin基因的3′臂、5′臂,其长度分别为1.4 kb、4.4 kb。将扩增的片段与T载体连接后进行部分序列测定,与已发表的Myostatin序列进行同源性比较,同源性为100%;对目的片段与载体进行酶切后定向连接形成转基因结构,经序列测定后,证明其插入方向和位置完全正确,构建了猪的Myostatin表达Neor、Tk基因的双标记转基因载体。     

猪源β-防御素2和3的研究进展

防御素是一类阳离子抗菌肽,广泛存在于动物和植物体内。猪源β-防御素2和β-防御素3能抵抗猪体内病原微生物,并能调节机体免疫功能,对生殖发育也有一定的生理作用。猪源β-防御素2能促进仔猪生长,对猪红细胞毒性低,能在毕赤酵母中高效表达,其转基因猪也具有较好的抗病性能。因此,猪防御素具有潜在的应用价值。本文就猪源β-防御素2和β-防御素3在体内的表达分布和调控因素、参与调节的信号通路、在生殖发育和畜牧养殖方面的新功能及应用潜力进行综述。     

Evaluation of use of human albumin in critically ill dogs: 73 cases (2003-2006)

OBJECTIVES: To evaluate the use of human albumin in critically ill dogs. Design-Retrospective case series. ANIMALS: 73 client-owned hospitalized dogs. PROCEDURES: Medical records of dogs that received human albumin were reviewed to assess effects of the use of human albumin on serum albumin concentration, colloid osmotic pressure, and total protein concentration; determine the relationships between these variables and outcome; and assess its safety. Data for signalment, diagnoses, physiologic variables, dosage, amount of crystalloid fluid administered prior to human albumin administration, complications, and outcome were reviewed. Additionally, pre- and postadministration values for serum albumin, colloid osmotic pressure, and total protein were recorded. RESULTS: Administration of human albumin resulted in significant changes in serum albumin, colloid osmotic pressure, and total protein. The serum albumin, total protein, degree of improvement in serum albumin, colloid osmotic pressure, and dosage of human albumin were significantly greater in survivors. Seventeen of 73 (23%) dogs had at least 1 complication that could be potentially associated with the administration of human albumin that occurred during or immediately following administration of human albumin. Three of 73 (4%) dogs had severe delayed complications. CONCLUSIONS AND CLINICAL RELEVANCE: Administration of human albumin significantly increased serum albumin, and total protein concentrations and colloid osmotic pressure, especially in survivors. Because of the high mortality rate of the study population and other confounding factors, it was uncertain whether complications were associated with the underlying disease or with human albumin administration. Acute and delayed complications may have been under-recognized.     

Effects of immune challenge on concentrations of serum insulin-like growth factor-I and growth performance in pigs

This study was designed to determine the long-term effects of repeated endotoxin treatment or immunization against human serum albumin on concentrations of serum insulin-like growth factor-I (IGF-I) and other indicators of growth performance in growing pigs. Thirty gilts (38.5 +/- 0.9 kg) were randomly assigned to 5 treatment groups (n = 6 animals/group): 1) lipopolysaccharide injections, 2) lipopolysaccharide pair-fed, 3) human serum albumin immunization, 4) human serum albumin pair-fed, and 5) control. Pigs in the lipopolysaccharide group were treated intramuscularly with lipopolysaccharide on Days 0-3. The pigs in the human serum albumin group were immunized with human serum albumin emulsified in Freund's adjuvant on Day 0 and administered a booster on Day 28. The lipopolysaccharide pair-fed pigs were matched by body weight and pair-wise fed with pigs treated with lipopolysaccharide. Similarly, human serum albumin pair-fed pigs were matched to human serum albumin immunized pigs. Serum IGF-I concentrations did not differ between or within groups. There was no difference in feed disappearance between groups prior to the initiation of treatments. The lipopolysaccharide group had a decrease (P = 0.013) in feed disappearance on Day 0 compared with control and human serum albumin groups. On Day 1, both lipopolysaccharide and human serum albumin groups differed (P < 0.05) from control. Average daily gain and total weight gain did not differ between groups; however, feed efficiency differed (P < 0.05) between lipopolysaccharide and control groups. Long-term effects of repeated endotoxin challenge or immunization on IGF-I concentrations and growth were not evident in the present study. This failure presumably was due to the development of endotoxin tolerance and a relatively innocuous vaccination against human serum albumin.     

Comparison of methods for depletion of albumin and IgG from equine serum

Background: Disease‐specific biomarkers hold diagnostic promise in both human and veterinary medicine, but serum biomarkers in low concentrations may be masked by the presence of abundant proteins, mostly albumin and IgG. Methods to deplete albumin and IgG exist, but efficacy of these methods for depleting equine serum of these proteins has not been established. Objective: The aim of this study was to determine if albumin and IgG could be depleted from equine serum using several commercially available kits and procedures. Methods: One‐dimensional gel electrophoresis followed by densitometry was used to determine percent of albumin, IgG, and both in pooled serum from 3 horses before and after application of 7 depletion methods. Repeatability was determined by applying the 2 best methods to serum samples from 6 grade horses. Results: For pooled serum, depletion rates varied from 35–90% for albumin and 0–94% for IgG. In the repeatability study, the ProteoExtract method combined with protein G Sepharose beads to remove additional IgG provided the best overall performance with 66% albumin depletion and 100% IgG depletion. A protocol using protein G Sepharose beads to remove IgG followed by ethanol precipitation of nonalbumin proteins with albumin remaining in the supernatant was the second most effective, with 85% albumin depletion and 55% IgG depletion. Although a multiprotein immunodepletion column effectively removed 90% of the albumin, the method was ineffective at removing IgG. Conclusion: Albumin and IgG removal kits optimized for human use have variable efficacy for equine serum. Combined use of the ProteoExtract kit and manual incubation with protein G Sepharose beads provided the most effective depletion.     

Enrichment of low-abundance proteins from bovine and porcine serum samples for proteomic studies

One of the main applications of serum proteomics is the identification of new biomarkers for animal disease or animal production. However, potential obstacles to these studies are the poor performance of affinity serum depletion methods based on human antigens when using animal samples, and loss of minor serum components bound to albumin and other proteins. In the present study, we have analyzed the efficiency and reproducibility of the ProteoMiner® beads with bovine and porcine serum samples, and compared to a traditional immunoaffinity-based albumin and IgG depletion system specific for human samples. The ProteoMiner kit is based on the use of a combinatorial peptide binding library and intends to enrich low-abundance proteins.     

Enrichment of low-abundance proteins from bovine and porcine serum samples for proteomic studies

One of the main applications of serum proteomics is the identification of new biomarkers for animal disease or animal production. However, potential obstacles to these studies are the poor performance of affinity serum depletion methods based on human antigens when using animal samples, and loss of minor serum components bound to albumin and other proteins. In the present study, we have analyzed the efficiency and reproducibility of the ProteoMiner® beads with bovine and porcine serum samples, and compared to a traditional immunoaffinity-based albumin and IgG depletion system specific for human samples. The ProteoMiner kit is based on the use of a combinatorial peptide binding library and intends to enrich low-abundance proteins.     

Adverse reactions suggestive of type III hypersensitivity in six healthy dogs given human albumin

CASE DESCRIPTION:6 healthy dogs given human albumin solution as part of a study were examined following development of an immediate hypersensitivity reaction (1 dog) and signs suggestive of a type III hypersensitivity reaction (all 6 dogs). CLINICAL FINDINGS: All 6 dogs were healthy prior to administration of human albumin solution. One dog developed signs of an immediate hypersensitivity reaction, characterized by vomiting and facial edema, during administration of human albumin solution. All 6 dogs developed signs of a delayed adverse reaction 5 to 13 days after administration of human albumin solution. Initial clinical signs included lethargy, lameness, edema, cutaneous lesions indicative of vasculitis, vomiting, and inappetance. TREATMENT AND OUTCOME: In the dog with signs of immediate hypersensitivity, signs resolved after administration of human albumin solution was discontinued and diphenhydramine was administered. Supportive treatment was provided after dogs developed signs of a delayed adverse reaction. Four dogs recovered, but 2 dogs died despite treatment. All 6 dogs were found to have antihuman albumin antibodies. There was no evidence of contamination of the human albumin solution. CLINICAL RELEVANCE: Findings suggest that administration of human albumin solution in healthy dogs with normal serum albumin concentrations may result in signs of a type III hypersensitivity reaction.     

A retrospective analysis of 25% human serum albumin supplementation in hypoalbuminemic dogs with septic peritonitis

This study describes the influence of 25% human serum albumin (HSA) supplementation on serum albumin level, total protein (TP), colloid osmotic pressure (COP), hospital stay, and survival in dogs with septic peritonitis. Records of 39 dogs with septic peritonitis were evaluated. In the HSA group, initial and post-transfusion TP, albumin, COP, and HSA dose were recorded. In the non-supplemented group, repeated values of TP, albumin, and COP were recorded over their hospitalization. Eighteen dogs survived (53.8% mortality). Repeat albumin values were higher in survivors (mean 23.9 g/L) and elevated repeat albumin values were associated with HSA supplementation. Repeat albumin and TP were higher in the HSA supplemented group (mean 24 g/L and 51.9 g/L, respectively) and their COP increased by 5.8 mmHg. Length of hospitalization was not affected. Twenty-five percent HSA increases albumin, TP, and COP in canine patients with septic peritonitis. Higher postoperative albumin levels are associated with survival.     

The role of albumin replacement in the critically ill veterinary patient

Objective: To review the human and veterinary literature on the physiological role and effects of therapeutic albumin supplementation. Data sources: Data from human and veterinary literature was reviewed. Human data synthesis: Hypoalbuminemia often occurs in a variety of critical illnesses, and contributes to the development of life‐threatening complications, including pulmonary edema, delayed wound healing, feeding intolerance, hypercoaguability, and multiple organ dysfunction. Serum albumin concentration has been used as a prognostic indicator in cases of chronic hypoalbuminemia. The use of albumin replacement therapy in humans is sometimes controversial, but may be associated with improved morbidity and decreased mortality. Veterinary data synthesis: Unlike human literature, there is a paucity of controlled clinical studies in the literature regarding albumin supplementation in veterinary patients. Rather, the majority of published studies were performed in experimental animals or via retrospective analyses. One recent study evaluated the use of plasma to improve albumin concentration in dogs with hypoalbuminemia. Other older studies investigated wound healing in dogs with experimentally induced hypoalbuminemia. As in human medicine, serum albumin concentration may be helpful as a prognostic indicator in critically ill dogs. Conclusion: Albumin is one of the most important proteins in the body because of its role in maintenance of colloid oncotic pressure, substrate transport, buffering capacity, as a mediator of coagulation and wound healing, and free‐radical scavenging. Albumin replacement in veterinary medicine is difficult, but until prospective clinical trials determine the efficacy of albumin replacement are conducted, a suggested clinical guideline would be to maintain albumin concentration at or above 2.0 g/dl utilizing fresh frozen plasma.     

人血清白蛋白载体的构建及其在山羊乳腺中的瞬时表达

根据GeneBank中人血清白蛋白(HSA)的基因序列,设计一对特异性的引物,从人肝cDNA文库中扩增出其编码序列,并加入酶切位点XhoⅠ,以pCDNA 3.1为模板,设计特异性的引物,并在上游引物加入NotⅠ,下游加入ApaⅠ和NotⅠ,扩增出筛选标记新霉素抗性基因neo,然后再以pBC1为基本骨架,在其NotⅠ、XhoⅠ位点处分别插入hsa和neo,从而得到乳腺表达载体pBC-NEO-HSA,将该质粒注射入奶山羊乳腺中进行暂时性的表达,结果显示所构建的载体能够有效地指导目的基因的表达,从而为下一步构建细胞系、克隆动物作好基础。     

A Comparison Between the Active Cutaneous Arthus Reaction and Immune-mediated Enteroluminal Neutrophil Emigration in Pigs

The induction of neutrophil emigration into the intestinal lumen in bovine serum albumin immune and nonimmune pigs by mucosal exposure to bovine serum albumin was studied using a ligated intestinal loop technique. In order to compare the response in the skin to that in the intestine, test materials were inoculated intracutaneously as well as enterally. Several histochemical procedures were applied to the intestinal mucosa and skin for evaluation of responses.

In immune animals, mucosal exposure to bovine serum albumin evoked the emigration of neutrophils into the intestinal mucosa and lumen. The neutrophil emigration tended to occur focally. Denudation of a few epithelial cells occurred at emigration sites. Hemorrhage, thrombosis, and edema, quite obvious after intracutaneous inoculation were not apparent after enteroluminal inoculation of bovine serum albumin into immune animals.

Enteroluminal inoculation of bovine serum albumin or bovine serum albumin plus anti-bovine serum albumin into nonimmune animals did not elicit neutrophil emigration or any other pathological lesion in the intestine, whereas intracutaneous inoculation of bovine serum albumin plus anti-bovine serum albumin into the same animals elicited an Arthus reaction.