山羊重组促卵泡素长效类似物基因在毕赤酵母中表达

为了获得长效FSH制剂, 利用重叠PCR技术将山羊FSH的a, b亚单位, 通过hCGb亚单位羧基末端延长肽(CTP)基因序列的连接, 构建成单链长效类似物基因FSHb-CTP-a。将其克隆至表达载体pPIC9K, 重组载体线性化处理后电转化至毕赤酵母GS115中, 经判型和G418筛选后获得高拷贝菌株His+Mut+。经甲醇诱导表达后, 进行SDS-PAGE和Western blot分析表明: 重组FSHb-CTP-a的转化子可以正确有效地表达目的蛋白, 分子量约为29 kD。放射免疫分析法(RIA)测定表达上清, 高拷贝转化子的平均表达量为91.849 mIU/mL, 显著高于低拷贝转化子的平均37.419 mIU/mL的表达量, 为FSH的结构研究和长效FSH制剂的生产奠定了基础。     

增加植酸酶基因appA-m的拷贝提高其在巴斯德毕赤酵母的表达量

为了进一步提高植酸酶的发酵效价,降低植酸酶生产成本,对毕赤酵母表达载体pGAPZα-A进行了改造。将表达载体pPIC9的AOX1启动子序列引入pGAPZα-A,使之成为甲醇可诱导型表达载体pAOXZα,插入植酸酶基因appA-m后得到重组载体pAOXZα-appA-m。以染色体上带有一个拷贝的appA-m基因、发酵效价可达到7.5×106IU/mL发酵液的重组酵母菌株74#为受体菌进行转化,在该重组菌株的染色体上的另一位点整合含有植酸酶基因的表达盒,经筛选到高表达植酸酶的重组子。通过PCR进行验证,植酸酶基因被整合到重组酵母的染色体上,且受体菌中原有的植酸酶基因结构未改变。重组菌在5L发酵罐经甲醇诱导120h植酸酶蛋白表达量达到4mg/mL发酵液,酶活性(发酵效价)达到1.2×107IU/mL发酵液以上,较含单拷贝植酸酶基因的受体菌株表达量有较大程度提高。PCR检测及表达量分析证明改良的菌株具有很好的遗传稳定性和表达稳定性。     

FAD依赖的葡萄糖脱氢酶多拷贝毕赤酵母菌株的构建及高效表达

通过体外多拷贝构建实现FAD依赖的葡萄糖脱氢酶(FAD-GDH)在毕赤酵母(Pichia pastoris)X33菌株中的高效表达。将前期构建的经密码子偏好性优化FAD-GDH基因插入到pPICZαA质粒中，通过同尾酶法酶切酶连构建含1～4个表达盒的重组表达质粒，分别电转至毕赤酵母X33中，成功筛选到各种重组菌株。qRT-PCR测定结果表明，载体所含的表达盒数目与嵌合进毕赤酵母基因组中的GDH基因拷贝数之间存在正相关关系。重组菌在试管水平用甲醇诱导72 h,酶活达到最高，其中4拷贝的转化子表达水平最高；选择1拷贝和4拷贝转化子进行10 L发酵罐扩大培养，1拷贝菌株诱导108 h酶活达到最高697.125 U/mL,4拷贝菌株诱导132 h酶活达到最高1 063.279 U/mL,比1拷贝酶活提高52.52%。结果表明通过增加目的基因拷贝数策略有助于提高FAD-GDH的表达量，为其进一步扩大生产提供参考。     

增加植酸酶基因appA-m的拷贝提高其在巴斯德毕赤酵母的表达量

为了进一步提高植酸酶的发酵效价,降低植酸酶生产成本,对毕赤酵母表达载体pGAPZα-A进行了改造。将表达载体pPIC9的AOX1启动子序列引入pGAPZα-A,使之成为甲醇可诱导型表达载体pAOXZα, 插入植酸酶基因appA-m后得到重组载体pAOXZα-appA-m 。以染色体上带有一个拷贝的appA-m基因、发酵效价可达到7.5×10⁶IU/mL发酵液的重组酵母菌株74#为受体菌进行转化,在该重组菌株的染色体上的另一位点整合含有植酸酶基因的表达盒,经筛选到高表达植酸酶的重组子。通过PCR进行验证,植酸酶基因被整合到重组酵母的染色体上,且受体菌中原有的植酸酶基因结构未改变。重组菌在5L发酵罐经甲醇诱导120h植酸酶蛋白表达量达到4mg/mL发酵液, 酶活性(发酵效价)达到1.2×10⁷IU/mL发酵液以上, 较含单拷贝植酸酶基因的受体菌株表达量有较大程度提高。PCR检测及表达量分析证明改良的菌株具有很好的遗传稳定性和表达稳定性。     

猪瘟病毒E2蛋白A/D抗原区基因在酵母中的分泌表达与鉴定

基于猪瘟病毒主要保护性抗原E2囊膜糖蛋白有两个相对独立的抗原结构单位-B/C抗原区和A/D抗原区,设计一对特异性的引物扩增猪瘟病毒E2蛋白的A/D抗原区基因,并将PCR产物克隆入含有强启动子PAox1和α-MF信号肽序列的巴斯德毕赤酵母表达载体pPICZαC中,构建成重组质粒pPICZα-AD,酶切线性化后电穿孔导入巴斯德毕赤酵母X33菌中,经ZeocinTM筛选得到5株高拷贝转化子,甲醇诱导表达.SDS-PAGE和Westernblot试验表明酵母培养上清液中含有具有良好反应原性的E2蛋白,蛋白表达量达175.8μg/mL.N-糖基化分析显示该表达蛋白在分泌过程中发生糖基化.该研究为研制防治猪瘟的亚单位疫苗与诊断试剂盒奠定基础.     

长效山羊促卵泡素类似物真核表达载体的构建及其在CHO细胞内的稳定表达

通过PCR重叠延伸法将人绒毛膜促性腺激素（hCG）的羧基末端延长肽（CTP）基因连接至山羊促卵泡素（FSH）15亚基的羧基末端,克隆入双表达载体pVITRO,测序证实后将表达载体转染CHO细胞,用潮霉素B筛选稳定表达的CHO细胞株。结果表明获得了长效山羊FSH基因,并得到稳定表达的CHO细胞株,表达量为0．105mU／mL,为进一步研究CTP结构与功能的关系,以及长效激素的理化特性奠定了基础。     

猪瘟病毒E2蛋白A／D抗原区基因在酵母中的分泌表达与鉴定

基于猪瘟病毒主要保护性抗原E2囊膜糖蛋白有两个相对独立的抗原结构单位-B／C抗原区和A／D抗原区,设计一对特异性的引物扩增猪瘟病毒E2蛋白的A／D抗原区基因,并将PCR产物克隆入含有强启动子PAOX1和α-MF信号肽序列的巴斯德毕赤酵母表达载体pPICZαC中,构建成重组质粒pPICZα-AD,酶切线性化后电穿孔导入巴斯德毕赤酵母X33菌中,经Zeocin^TM筛选得到5株高拷贝转化子,甲醇诱导表达。SDS-PAGE和Western blot试验表明酵母培养上清液中含有具有良好反应原性的E2蛋白,蛋白表达量达175．8μg／mL。N-糖基化分析显示该表达蛋白在分泌过程中发生糖基化。该研究为研制防治猪瘟的亚单位疫苗与诊断试剂盒奠定基础。     

一种获取巴斯德毕赤酵母高拷贝重组子的新方法

目的：探讨一种简化的高效获取用于表达重组蛋白的巴斯德毕赤酵母高拷贝重组子的新方法,即多重转化筛选法。方法：按经典方法转化宿主菌和G418多拷贝筛选法从转化子中筛选出含目的的基因表达盒拷贝数最高的菌株,然后以之作为新一轮的转化宿主菌。如此重复进行数轮上述化筛选过程,直到获得含有尽可能多拷贝目的的基因表达盒的重组子;同时将此方法,即多重转化筛选法与经典方法进行比较。结果：与经典方法相比,多重转化筛选法通过筛选较少量转化子即可轻而易举地获得较多的高拷贝重组子,工作量更小,效率更高。结论：多重转化筛选法是一种更高效地获取毕赤酵母高拷贝重组子的新方法。     

长效人促卵泡激素CHO细胞株的构建及其体内活性

促卵泡激素(FSH)是具有促进卵泡与睾丸发育作用的一种垂体糖蛋白激素。因其体内半衰期较短,临床上需要连续10 d以上每天注射,病人使用非常不方便。本文旨在通过提高糖基化程度,研制一种长效FSH。通过一段含有两个N-糖基化位点的连接序列,将人FSHα链与β链cDNA融合,并插入pcDNA3.1(+)表达载体。表达载体转染CHO-K1细胞后,通过G418筛选得到阳性单克隆细胞,并经PCR和Western blotting证实。该重组FSH为单链蛋白,分子量约为49 kDa。经无血清培养,工程细胞株培养上清液中重组FSH的表达量可达3 mg/L。单次注射该重组FSH能够促进大鼠卵巢发育与卵泡成熟,且药效与连续8次注射Folltropin-V的相近。实验结果显示,本研究已成功获得一种长效重组FSH。     

猪瘟病毒E2蛋白B/C抗原区基因在毕赤酵母中的表达与鉴定

基于猪瘟病毒主要保护性抗原---E2囊膜糖蛋白有两个相对独立的抗原结构单位---B/C抗原区和A/D抗原区 ,设计引物扩增编码猪瘟病毒E2蛋白B/C抗原区的基因 ,将大小为261bp的PCR产物插入含有强启动子P_AOX1和α-MF信号肽序列的巴斯德毕赤酵母 (Pichiapastoris)表达载体pPICZαC中 ,构建成重组质粒pPICZα-BC ,酶切线性化后电穿孔导入巴斯德毕赤酵母菌X₃₃ 中 ,经Zeocin^TM 筛选得到 3株高拷贝转化子 ,甲醇诱导表达 ,SDS-PAGE和Westernblot及ELISA试验表明 ,酵母培养上清液中含有具有良好反应原性的E2蛋白。为研究亚单位疫苗或诊断抗原打下坚实基础。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

肝癌中HBV和HCV基因和抗原的分布及意义

采用原位分子杂交方法检测HCV RNA及HBV X基因;采用免疫组织化学方法研究HCV核心抗原,非结构区C33c抗原及HBxAg在肝细胞肝癌中的定位及分布.结果表明(1)HCV RNA、HBV X基因在肝细胞肝癌组织检出率分别为40%(55/136)和82%(112/136).HCV RNA定位于癌细胞的胞浆内,阳性细胞呈散在、灶状及弥漫分布三种形式;HBV X基因在肝癌细胞中的分布呈胞浆型、核型及核浆型,阳性细胞也呈上述三种分布形式;(2)HCV C33c抗原、核心抗原在肝细胞肝癌中的阳性率为81%(133/164)及86%(141/164).C33c抗原定位于癌细胞及肝细胞的胞浆内;核心抗原既定位于癌细胞核中,又可定位于胞浆中.C33c抗原阳性细胞以灶状分布为主;而核心抗原阳性细