H6N6亚型禽流感病毒N6基因的原核表达与多克隆抗体制备

为表达H6N6亚型禽流感病毒（avian influenza virus,AIV）神经氨酸酶（NA）蛋白,并制备多克隆抗体,本试验根据GenBank中AIV N6基因序列（登录号：MG434500）设计特异性引物,对贵州地区分离的H6N6亚型AIV贵州株进行N6基因PCR扩增,将其克隆到原核表达载体pET-32a （+）中,构建重组原核表达载体pET-32a-N6,转化大肠杆菌BL21（DE3）感受态细胞,经IPTG诱导表达His-N6重组蛋白,将诱导产物超声破碎离心后,利用镍柱对重组蛋白进行纯化,并利用SDS-PAGE和Western blotting对表达蛋白进行双重鉴定,将纯化后的重组蛋白免疫新西兰大白兔制备多克隆抗体,并通过间接ELISA检测其效价。结果显示,AIV贵州株N6基因编码区长1 380 bp,可编码459个氨基酸,其中175－207 bp缺失33个核苷酸;重组质粒pET-32a-N6经双酶切鉴定分别获得大小为5 900 bp左右的载体条带和1 380 bp左右的目的基因条带,成功构建了pET-32a-N6重组质粒;蛋白超声破碎后经SDS-PAGE发现,重组蛋白主要存在于沉淀中,以包涵体形式存在,蛋白分子质量约为70 ku,与预期结果一致;纯化后的重组蛋白经SDS-PAGE和Western blotting双重鉴定,均在70 ku处出现条带,说明纯化的蛋白为重组蛋白pET-32a-N6,表达产物具有免疫学活性,包涵体经变性、复性处理,重组表达蛋白分别被His抗体和兔源抗N6多克隆抗体所识别。间接ELISA检测其效价高于1∶3 200。以上结果表明,试验成功克隆、表达了H6N6亚型AIV的N6基因,所制备的N6蛋白多克隆抗体具有良好的免疫活性,能被His抗体和兔源抗N6多克隆抗体所识别。     

H6N6亚型禽流感病毒反向遗传操作系统的建立

为建立H6N6亚型禽流感病-毒(AIV) A/Chicken/GuangDong/S1311/10 (W-CKGD)反向遗传操作系统,本研究构建了W-CKGD的8个重组质粒,拯救出AIV A/Chicken/GuangDong/S1311/10 (R-CKGD).全基因序列测定结果表明,救获病毒R-CKGD与野生病毒W-CKGD的核苷酸序列完全相同.R-CKGD与W-CKGD具有相同的受体结合特性和对BALB/c小鼠均呈低致病性,以106 EID50剂量鼻腔感染BALB/c小鼠,病毒仅在小鼠的呼吸道复制,可以引起小鼠体重下降但均不造成死亡.实验结果表明,R-CKGD与W-CKGD保持了一致的生物学特性,从而为进一步研究H6亚型AIV的致病机理及跨宿主传播机制等提供了良好的平台.     

黄芩苷对H6N6禽流感患鼠血液指标的影响

为了研究黄芩苷对H6N6禽流感(avian influenza,AI)病毒感染小鼠的血液学指标的影响。选用6-8周龄小鼠108只,随机平均分为阴性对照组、病毒阳性组、低、中、高黄芩苷治疗组和西药对照组;攻毒:阴性对照组尾静脉注射灭菌生理盐水0.2mL/只,其余各组均尾静脉注射H6N6禽流感病毒(108EID50) 0.2mL/只;治疗:攻毒3 d后进行灌胃给药,黄芩苷治疗组(0.014,0.028,0.056 mg/只/d),西药对照组(0.39 mg/只/d),病毒阳性组、阴性对照组每天给予等体积灭菌生理盐水,分别于治疗后3、6、9 d采血进行血常规检测。通过对各组别患鼠血液中白细胞、红细胞和血小板数目变化的分析。结果表明:(1)治疗后3、6、9 d高剂量黄芩苷组白细胞总数、中性粒细胞数目均极显著低于病毒阳性组和低、中剂量黄芩苷组(P0.01);(2)在治疗3、6和9 d时淋巴细胞数目和淋巴细胞百分比均极显著或显著高于病毒阳性组、低剂量黄芩苷治疗组和中剂量黄芩苷治疗组(P0.01或P0.05),与西药对照组差异不显著(P0.05);(3)在治疗3、6和9 d时,所有黄芩苷治疗组红细胞数目、血红蛋白浓度和平均红细胞血红蛋白含量均极显著高于病毒阳性组(P0.01),与阴性对照组合西药对照组差异不显著(P0.05);(4)在治疗3、6和9 d时所有黄芩苷治疗组血小板数目均极显著低于病毒阳性组(P0.01)。小结:黄芩苷在较高浓度时,对H6N6禽流感引起的炎症反应,具有降低白细胞总数,抑制出血反应的作用。     

Generation of monoclonal antibodies to porcine interleukin 6 (PIL-6) using the recombinant PIL-6 expressed in Escherichia coli

Porcine interleukin-6 (PIL-6) protein without signal peptide was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli. The fusion protein was expressed in an insoluble fraction, however, it was solubilized by refolding procedure using urea. From the solubilized protein, the recombinant PIL-6 (rPIL-6) was purified by a batch method using glutathione sepharose 4B and PreScission protease cleavage. By the B3B1 hybridoma cell proliferation assay, biological activity of the purified rPIL-6 was confirmed. Three monoclonal antibodies (MAbs) named 2B-1, 5A-8 and 4C-3 were generated by using the rPIL-6 as an immunogen. Immunoglobulin isotypes of the MAbs were IgG2a (4C-3) and IgG2b (2B-1 and 5A-8). For the epitope analysis, additive enzyme-linked immunosorbent assay and immunoblot analysis using deletion mutants of PIL-6 were performed. These experiments revealed that the two MAbs (2B-1 and 5A-8) recognize an overlapped epitope and the other (4C-3) recognizes a distinct epitope, and all epitopes reside in the region of aa26-64 of PIL-6.     

一株三穗鸭源H6N6亚型禽流感病毒的分离鉴定

探讨H6N6亚型禽流感病毒在鸭群中的流行状况,为贵州省流感疫情动态监测及防控提供依据。从贵州三穗县某养鸭殖场采集50份泄殖腔棉拭子,处理后经过绒毛尿囊腔接种9日龄非免疫鸡胚,通过血凝(HA)和血凝抑制试验(HI)分离到1株既不是新城疫病毒,也不是H5、H7和H9亚型禽流感的病毒;提取病毒RNA,通过禽流感病毒HA和NA基因通用引物扩增得到相应的HA和NA基因片段。经过测序比对分析证实所分离病毒为H6N6亚型禽流感病毒。贵州鸭群中存在H6N6亚型禽流感病毒,为进一步研究其起源和致病性提供了重要依据。     

鸡白细胞介素-6的原核表达及其对疫苗免疫增强效果的研究

本研究采用PCR技术,扩增、克隆了鸡白细胞介素-6（ChIL-6）成熟肽基因并进行原核表达,对表达的鸡白细胞介素-6组蛋白（rChIL-6）通过尿素变性、复性液复性、PBS溶液透析等步骤进行纯化,经测定rChIL-6的促小鼠脾脏淋巴母细胞的增殖能力来评估重组蛋白的生物学活性;用4个不同浓度的rChIL-6在不同部位与NDV活疫苗、AIVH5N1灭活疫苗同时肌肉注射来评价其免疫增强作用。研究结果表明,表达rChIL-6为25ku左右,经纯化后的蛋白纯度在95%以上;100pg/mLrChIL-6注射组具有较高的促小鼠脾淋巴母细胞增殖活性;不同浓度的rChIL-6注射组对NDV和AIVH5N1疫苗都具有较好的免疫增强作用,但对其免疫抗体产生时间及抗体滴度峰值出现和维持时间均没有明显的影响,其中0.03μg/mLrChIL-6浓度注射组的对鸡疫苗的免疫增强作用最明显,过高或过低浓度的rChIL-6对疫苗免疫增强作用不显著。这些研究为新型免疫增强佐剂开发以及鸡免疫失败性疫病的预防和治疗奠定了基础。     

On The Normal Activities of Glucose-6-Phosphate Dehydrogenase and 6-Phospho-Gluconate Dehydrogenase in Bovine Erythrocytes

The activities of glucose-6-phosphate dehydrogenase and of 6-phos-pho-gluconate dehydrogenase have been determined in cows, heifers, bulls and calves. The material consisted of 80 RDM, 46 SDM, 31 Jersey animals older than 3 months, 16 newborn RDM calves and 29 fetuses of various breeds.

1. In RDM averages of 322 ± 56 units G6PD and 52.8 ± 12.2 units 6PGD per 100 ml PGV were found. In SDM 330 ± 43 units G6PD and 47.1 ± 9.2 units 6PGD were found, and in Jersey 315 ± 39 units G6PD and 49.2 ± 8.0 units 6PGD per 100 ml PGV. The activities of both dehydrogenases were shown to be normally distributed in all three breeds.
2. The 6PGD activities in SDM were significantly lower than in RDM, but no other breed differences between the mean activities were detected.
3. There is no relation between the age (apart from fetuses and newborn calves), and milk yield of the animals and the activities of G6PD and 6PGD in the erythrocytes.
4. Statistical treatment of the results demonstrates that there is no relation between the G6PD and 6PGD activities in the blood samples, and that the enzyme activities in units per 100 ml PGV is not correlated with MCHG.
5. Further, the statistical treatment shows that when measuring G6PD and 6PGD activities in erythrocytes, PGV is to be preferred to hemoglobin concentration as parameter.
6. In bovine fetuses G6DP activities more than twice as high as in adult erythrocytes were found, while the 6PGD activities were only slightly higher than in adult erythrocytes. After birth the high G6PD activities decreased over a period of 40 days to the normal level for mature cattle. The 6PGD activity also attains the normal level for older animals in approximately 40 days after birth.

     

Detection of mycoplasmas in cell cultures, using L6M, containing 6-methylpurine deoxyriboside

Methods for the detection of mycoplasmas in cell cultures with 6MPDR are rapid and inexpensive and do not require special skill in the mycoplasma culturing. Properties of L6M, a dry, lyophilised, sterilised, and thermostabile reagent containing 6MPDR, are described. Its diagnostic value is comparable to that of the liquid, 6MPDR containing reagent MycoTect requiring storage at -70 degrees C.     

黄芩苷对H6N6禽流感感染小鼠肺损伤的影响

为探究黄芩苷对H6N6禽流感病毒感染小鼠肺损伤的影响，选取108只昆明小鼠，随机平均分为6组(对照组、阳性组、西药组和低、中、高剂量黄芩苷组)。攻毒3 d后用不同剂量黄芩苷连续治疗9 d,各组在第3,6,9天时随机选6只小鼠，取其肺脏进行病理观察及HE染色制作切片，检测肺组织病毒含量及小鼠血清中IL-1、TNF-α、IFN-γ和5-HT含量。结果表明：攻毒3 d后，相比对照组，其余各组小鼠肺脏出现病理损伤；治疗3 d时，相比阳性组，高剂量黄芩苷组小鼠肺脏指数、病理评分和病毒含量均显著降低(P<0.05),治疗6 d时，相比阳性组，低、中、高剂量黄芩苷组小鼠血清中IL-1、TNF-α、5-HT含量均极显著降低(P<0.01),中、高剂量黄芩苷组IFN-γ均极显著升高(P<0.01);相比西药组，高剂量黄芩苷组小鼠肺脏指数、病理评分、病毒含量及血清中IL-2含量均无显著差异(P>0.05)。说明黄芩苷通过降低病毒在肺脏中的复制，调节血清中炎性因子(IL-1、TNF-α、IFN-γ和5-HT)含量，减轻H6N6禽流感患鼠肺的损伤。     

香蕉枯萎病生防细菌ZJ6-6的盆栽防治效果及其生防基因分析

通过病原菌和生防菌人工接种方法测定生防细菌ZJ6-6对香蕉枯萎病的盆栽防治效果，扩增和序列分析ZJ6-6的生防基因。结果表明：ZJ6-6处理香蕉的萎缩指数和导管褪色指数比对照香蕉分别降低40.5%和43.9%，其对香蕉枯萎病具有显著防治效果；ZJ6-6至少含有6个生防基因ituD、lpa-14、bmyB、fenD、srfAA、srfAB，理论上具有合成脂肽抗生素的能力，这可能是其能防治香蕉枯萎病的原因。本研究结果对ZJ6-6合成分泌的抗菌物质鉴定及其生防机理研究具有重要意义。     

1株禽源猪流感H6N6病毒的反向遗传系统的建立

为建立禽源猪流感病毒A/swine/Guangdong/K6/2010(H6N6)的反向遗传系统,本试验构建了包含有GDK6毒株基因组的8个重组质粒,转染293T细胞后成功拯救出病毒r GDK6。救获病毒与亲本病毒拥有相同基因序列,其抗原性、经HI试验、TCID50试验和小鼠攻毒试验表明,救获病毒的生物学特性与对小鼠的致病性与亲本病毒一致。禽源猪流感H6N6病毒的反向遗传系统的成功建立为进一步研究H6亚型流感病毒的分子致病机理与病毒基因功能及新型疫苗创制提供了技术平台。     

Effect of 6-mercaptopurine on rat placenta

In order to investigate the toxic effects of 6-mercaptopurine (6-MP) on placental development, we examined sequential morphology in the placentas from rats exposed to 6-MP. 6-MP was intraperitoneally administered at 60 mg/kg during gestation days (GDs) 11 to 12, and the placentas were sampled on GD 13, 15 or 21. In the 6-MP-treated group, maternal body weight suppression, increased death embryo/fetus ratio and some malformations were observed. The placenta weights were decreased on GDs 15 and 21. Macroscopically, placentas on GD 21 were small, brittle and thin with a white peripheral rim. Histopathologically, in the labyrinth zone, 6-MP treatment mainly evoked decreased mitosis on GDs 13 and 15, increased apoptotic cell on GDs 13, 15 and 21 and thinning on GDs 15 and 21. In the basal zone, 6-MP evoked decreased mitosis on GDs 13, and PAS-positive material in the spongiotrophoblasts was still detected on GD 15. Thickening of the basal zone was observed with cytolysis of glycogen cells, apoptosis and an increased number of composed cells on GD 21. In conclusion, 6-MP administration in pregnant rats induced growth arrest of the labyrinth zone and developmental delay in the basal zone, leading to small placentas. The fetotoxicity of 6-MP may be responsible for its direct anti-proliferative effects and resulting placental dysfunction.     

利用EST数据库资源克隆家蚕Bombyx mori6-磷酸葡萄糖酸脱氢酶基因

以果蝇6-磷酸葡萄糖酸脱氢酶(6-[jps[jpg;icpmate dehydrogenase,6PGDH)基因cDNA序列为信息探针,对家蚕EST数据进行同源检索筛选,克隆到了家蚕6-磷酸葡萄糖酸脱氢酶基因的cD-NA序列,该基因全长2011bp.经RT-PCR克隆、序列分析验证,结果表明与电子克隆序列完全一致;该基因具有完整的开放阅读框(0RF),编码蛋白为483个氨基酸;通过与人、果蝇、家鼠、摇蚊、线虫的6PGDH蛋白序列比较,发现该基因具有高度的保守性.