人生长激素基因在花叶芋中的整合与表达

本试验将人生长激素(hGH)基因插入质粒载体pLGV1103中,构建了重组质粒pLB-9,将此重组质粒引入携带Ti质粒pGV3850的农杆菌,经同源重组,获得共整合重组体pGL198(hGH)Ti质粒。用叶盘共培养法,使pGL198(hGH)转化单子叶植物花叶芋(Caladium bicolor),获得转基因再生植株。经胭脂碱、新霉素磷酸转移酶、Southern印迹以及Western印迹等分析,表明hGH基因已整合到花叶芋DNA中,并且合成了分子量为22kD的表达产物。     

基因密码子扩展技术在生物医药领域的应用

自然界中的蛋白质由20种天然氨基酸组成.基因密码子扩展技术利用无义密码子实现蛋白质中非编码蛋白氨基酸(noncanonical amino acids, ncAAs)的定点引入.目前,该技术已在不同物种的体蛋白中插入了几百种ncAAs,其应用已经扩展到了生物医药领域.一方面其定点引入生物正交化学官能团的特性,已在蛋白与抗体制药领域展现出了强大的应用潜力;另一方面其实现对无义密码子的通读调控,拓展了由合成生物学引领的新一代生物医药的应用和发展.本文重点总结了近年来基于基因密码子扩展技术所开发的各类蛋白抗体及细胞类药物,以及基于这一技术引领的新型生物治疗方法.     

化学与酶促相结合合成并克隆核糖体失活蛋白Gelonin基因

利用已知Gelonin的氨基酸序，逆向推算出整个基因的碱基序列，根据E.coli的密码子偏爱性和后续基因克隆的需要，通过沉默突变设计相应的酶切位点和碱基序列，将整个基因分为四段，每个片段约175－220pb，每一个片段中的互补链从5′末端用化学合成100－120的碱基单链，其中两个链的3′末端有20个互补碱基。利用T4DNA聚合酶酶促添补成双链DNA，用分子克隆技术，分别构建重组子，然后再构建成含整个Gelonin基因的表达载体pET-gel进行表面，经诱导后，获得了一个28000的重组蛋白，并主要以可溶形式存在。     

SARS病毒核衣壳蛋白的表达与鉴定

依据Genebank中SARS基因组序列和酵母菌对密码子的选择性,采用人工合成的方法,合成了优化的SARS病毒核衣壳蛋白(N)的全基因(1296bp),与CTL表位基因(195bp)重组后,将其克隆到酵母分泌型表达载体pPIC9K中,构建成重组表达载体pPIC9K-N.重组载体转化毕赤酵母GS115,并经MD平板和MM平板筛选及PCR鉴定,得到阳性重组酵母工程菌GS115-pPIC9K-N.用甲醇诱导其分泌表达目的蛋白并对表达产物进行分析、浓缩与鉴定.结果表明,SARS病毒核衣壳蛋白能实现在毕赤酵母中高效表达,表达量达到20%,初步纯化后的产物具有良好的抗原特异性.     

启东鸭肝癌中人HBV突变体(pDKHBV)的DNA序列

本文首次报道对启东鸭肝癌中存在的人HBV样DNA~([3])(pDKHBV)的全序列分析。本工作建立了pDKHBV的M_(13)重组克隆及亚克隆,并应用末端终止法测定了它的全序列。结果表明,它长3218bp,与鸭乙型肝炎病毒无关,而同人乙型肝炎病毒(HBV)基因组有91％以上同源性(其中与adw_2亚型有99.0％同源性),存在与HBV一致的4个开放阅读框架,并在Pre-S_1基因下游59个核苷酸,以及C基因3′端分别有1个和2个核苷酸的缺失,造成Pre-S_1基因第58个密码子、C基因第158个密码子出现终止信号,以及P基因第21个密码子起发生移码突变。以上结果表明启东鸭肝癌中存在adw_2亚型HBV的非复制型突变体。由于启东鸭肝癌高发且与人肝癌高发有相关性,提示HBV作为人和鸭肝癌共同病因的可能性。     

外显子周期三行为特征的研究

基因中蛋白质的编码区具有周期三行为这一规律已成为目前基因预测的理论基础, 采用功率谱对外显子的周期三行为特征进行了研究, 结果表明: 大多数外显子独立存在于基因中时并不具有周期三行为, 而当基因被剪切后外显子连在一起编码蛋白质的时候才具有周期三行为. 并且这种行为特征与外显子的长度、碱基在密码子三个位置上的分布以及氨基酸密码子的使用偏好均有密切关系, 同时符合蛋白质翻译次序的外显子也具有对密码子使用的偏好性, 这一研究结果对于提高基因预测的准确率以及内含子功能的研究具有重要意义.     

人工合成杆状病毒后期启动子的探讨

本文以苜蓿丫纹夜蛾核型多角体病毒(Autographa californica Nuclear PolyhedrosisVirus,AcNPV)多角体、p10、病毒粒子衣壳和核心碱性蛋白启动子的保守序列为基础,设计合成杆状病毒后期启动子——92和96碱基的互补寡聚核苷酸,并以此重组出5株在多角体启动序列、翻译起始密码子ATG上游-92处,分别或同时插入反方向的合成和多角体启动子,以及氯霉素乙酰基转移酶(CAT)基因的AcNPV毒株,探讨了带这些启动子的CAT在草地夜蛾(Spodoptera frugiperda)细胞中的表达规律及多角体基因对其表达的影响。形成多角体的重组病毒株AcNPV-vⅪⅤⅥ~+CAT和AcNPV-vSⅪⅤⅥ~+CAT中,处于合成启动子与多角体ⅪⅤ启动顺序下游的CAT基因在细胞中的表达量,要比只在多角体启动子带动下高3倍。     

水稻组蛋白H3基因和1.5-二磷酸核酮糖羧化酶/加氧酶小亚基基因的克隆和结构分析

本文从水稻(Oryza sativa,IR26)细胞核中分离出的几个基因,测定了它们的DNA核苷酸序列,并从水稻IR26基因组文库中筛选和分离到一个14.8kb的DNA片段,其中含有一个H3基因和另一个类似H3的假基因。DNA序列分析的结果表明:水稻H3基因的编码顺序有405bp长,其5′和3′端的非编码区则含有几个与一般真核基因或组蛋白基因共同的调控序列。水稻H3基因的密码子有一显著特点,密码子的第三个核苷酸98％为G和C,通过Southern转移分析,表明水稻二倍体基因组中大约有50个左右H3基因,从同一个水稻基因文库中,我们还分离鉴定到了rbcS基因,它编码1,5-二磷酸核酮糖羧化酶/加氧酶(Rubisco)的小亚基,水稻rbcS基因的顺序含有一个内含子,其位置与小麦的相同,水稻和小麦rbcS基因所编码的转移肽,其最初18个氨基酸彼此是相同的。     

基因兴奋剂检测的现状与策略

本文综述了基因兴奋剂检测的现状和反基因兴奋剂的策略。归纳了可能被运动员滥用的基因兴奋剂,分析了由这些基因表达的促红细胞生成素(EPO)、人生长激素(hGH)等蛋白的检测进展,讨论了未来检测基因兴奋剂的可能策略。     

胰泌素基因的化学合成和克隆

本文报道的内容为胰泌素基因的化学合成和克隆。胰泌素为一由2了个氨基酸组成的肠胃道多肽激素,该多肽合成基因的顺序由计算机辅助设计,共216个核苷酸碱基;合成基因选用酵母细胞偏爱的密码子,并在其中设置了一些便于今后用于基因改造、表达调控和产物分离所需的位点,排除了可能影响酶促连接反应的各种重复顺序;用化学法(固相磷酸三酯法和固相亚磷酸三酯法)合成了12个基因片段,长度为12寡聚核苷酸至24寡聚核苷酸,聚丙烯酰胺凝胶电泳分离纯化合成产物;利用T_4DNA连接酶组建完整的胰泌素基因,克隆于pWR 13质粒中,构建了一个含胰泌素基因的新质粒pWS 1。     

Efficient two-step synthesis of methylphytylbenzoquinones: precursor intermediates in the biosynthesis of vitamin E

Methylphytylbenzoquinone was synthesized from δ-tocopherol by a simple two-step sequence. Oxidative cleavage of the benzopyran ring with cerium sulfate followed by dehydration using Burgess reagent afforded the methylphytylbenzoquinone as a mixture of positional and geometric isomers which were separated by HPLC. The biological activity of the product corresponds to the natural biosynthetic precursor of vitamin E. The above method is a general procedure applicable to the preparation of any of the tocopherol derivatives.     

Reaction of substituted alkynols with alkoxycarbene complexes of chromium: a facile synthesis of substituted α,β-unsaturated-γ-butyrolactones

Substituted α,β-unsaturated-γ-butyrolactones, were synthesized from Fischer chromium carbenes and substituted alkynols in a two-step sequence. This method demonstrates a novel and mild route for the synthesis of this class of molecules.     

Synthesis of Codon-optimized Human Interleukin-18 Gene by Combination of Chemical and Enzymatic Method

According to the amino acid sequence and codon preference of E. coli, the human interleukin-18（IL-18） gene was optimized to avoid the rare codons. The total length of the synthesized gene is 571 bp; 18 oligonucleotides, DNA fragments were designed and synthesized by the phosphoramidite four-step chemical method. The whole DNA sequence was synthesized by a one-step total gene synthesis method, and then inserted in pUC18 vector. Five positive clones identified by blue-white colony screening were sent to Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. for sequencing. The sequencing result shows that one clone contained the complete correct gene in all the five positive clones.     

Arenes to anilines and aryl ethers by sequential iridium-catalyzed borylation and copper-catalyzed coupling

[reaction: see text] N-Alkyl- and N-arylanilines were synthesized from arenes by a two-step sequence of iridium-catalyzed borylation and copper-catalyzed coupling with amines. Diaryl ethers were obtained by a related sequence of arene borylation, followed by coupling with phenols. In particular, 3,5-disubstituted arylamines and aryl ethers were prepared by initiating this sequence with meta-substituted arenes.     

3种新型聚苯基醚喹噁啉的合成

用4,4′-二氟代二苯甲酰与双酚A的溶液缩聚制得聚醚苯偶酰(PEB),经邻苯二胺改性后,通过两步法得到聚苯基醚喹噁啉（PEQ-1）。 在PEB的改性反应中加入质量分数分别为1.0%和1.3%的3,3′,4,4′-四胺基二苯醚,可有效增加聚苯基醚喹噁啉的相对分子质量,得到PEQ-2和PEQ-3（总收率≥90%）,其质均相对分子质量分别为7.492×104和1.486×105。 并用IR、 NMR等对这些聚合物进行了表征。 该法操作简便,具有潜在的应用价值。     

Diversity-oriented synthesis of polycyclic diazinic scaffolds

An efficient and versatile synthesis of a polycyclic diazinic system starting from oxazine has been developed using a two-step Michael/retro Michael and cyclization sequence. The substrates were synthesized with good to high yields giving rapid access to molecular diversity.     

Photodecarboxylative addition of carboxylates to phthalimides: a concise access to biologically active 3-(alkyl and aryl)methylene-1H-isoindolin-1-ones

A series of 3-(alkyl and aryl)methyleneisoindolin-1-one derivatives were synthesized in a simple two-step procedure using a recently established photodecarboxylative addition of carboxylates to phthalimides as the key-step. Subsequent acid-catalyzed dehydration and deprotection furnished the desired target compounds with high E-selectivity. The reaction sequence was applied to the synthesis of the known bioactive phenylethylene derivative, AKS-186. Different analogues, including heteroatom-containing isosteres were also synthesized using this approach.     

Nazarov cyclisation of dienone-esters and tetrahydropyrones using trimethylsilyltriflate

Dienone- and tetrahydropyrone- esters undergo Nazarov-type cyclisation using trimethylsilyltriflate.

Cyclopentenone esters have been synthesized via a Nazarov cyclisation of the corresponding ,′-dienone esters or tetrahydro-γ-pyrone esters employing trimethylsilyltriflate at room temperature. The dienone esters were synthesised by a short two-step acylation-Knoevenagel sequence.     

Synthesis of C3-substituted 4-azaindoles: an easy access to 4-azamelatonin and protected 4-azatryptophan

C3-Substituted-4-azaindoles were synthesized from pyridylacetonitriles in a two-step sequence allowing the easy introduction of a range of substituents. This strategy permits the rapid synthesis of 4-azamelatonin and a protected 4-azatryptophan.     

一步法合成异长叶烷酮及其衍生物

以长叶烯为原料，一步合成异长叶烷酮，产率接近分步合成法。制备了异长叶烷酮的两种异构体及其衍生物。鉴定了它们的香型。