新型兽用疫苗的研究进展

新型兽用疫苗是免疫学和病毒学的研究热点,主要包括亚单位疫苗、基因工程缺失苗、重组病毒活载体疫苗和基因疫苗。上前已经被广泛的应用于研究和疫病的防治上。最近的疫苗发展方向集中于在基因疫苗上。高效、安全、稳定、成本低是疫苗开发的重点。本文介绍了新型疫苗的现状,以及今后发展的趋势和策略。     

基因打靶技术的研究进展

基因打靶技术是一项新兴的分子生物学技术,是利用外源DNA与受体细胞染色体DNA上的同源序列之间发生重组,并整合在预定位点上,从而改变细胞遗传特性的方法。它的产生是遗传工程领域的一次革命,为发育生物学、分子遗传学、免疫学及医学等学科提供了一个全新的、强有力的研究手段。目前基因打靶技术在研究基因的结构和功能、表达与调控,转基因及基因治疗等方面均取得了进展。但基因打靶技术仍存在一些问题,主要是打靶的效率太低。本文综述了基因打靶技术的原理、操作程序并对提高基因打靶效率的可能途径进行了探讨。 Progress on Gene Targeting LIU Hong-quan1,DAI Ji-xun1,YU Wen-gong2,YANG Kun-feng1 1.Ocean University of Qingdao,College of Marine Life Sciences,Qingdao 266003,China; 2.Institute of Marine Drugs and Foods,Qingdao 266003,China Abstract:Gene targeting is a rising technology in molecular biology,which is defined as the introduction of exogeneous DNA to specific site in genome by homologous recombination,and consequently change the hereditary character of the cell.This technology provides a new and powerful means for research in developmental biology,molecular genetics,immunology and medicine.Progresses have been made in exploring gene structure and function,gene expression and regulation,transgene and gene therapy with the application of gene targeting.But there are some problems in gene targeting,especially for the low efficiency.This article just provided a review of the principle and program of gene targeting,and discussed the possible approaches to increase the efficiency of gene targeting. Key words：gene targeting;homologous recombination;targeting vector;targeting efficiency     

Proviral genomic sequence analysis of Chinese donkey leukocyte attenuated equine infectious anemia virus vaccine and its parental virus strain Liaoning

Proviral DNA was extracted from donkey leukocyte infected with Chinese donkey leukocyte attenuated equine infectious anemia virus (DLA-EIAV), and peripheral blood lymphocytes (PBL) from a horse infected with the virulent EIAV strain Liaoning (EIAV L). The entire proviral DNA from both viruses was cloned and sequenced. The lengths of complete genomic sequences of DLA-EIAV and EIAV L provirus were 8266 bp and 8235 bp, respectively. Sequence comparison indicated that DLA-EIAV shares 97.0% and 97.5% in sequence homology with EIAV L and donkey-adapted EIAV (DA-EIAV), respectively. Lots of variations occurred in long terminal repeat (LTR, consisting of U3, R, U5), ORF S2, and envregions between DLA-EIAV and EIAV L. The nucleotide sequence differences of the two viruses in U3, R, U5, ORF S2, and env are 13.2%, 7.5%, 5.1%, 3.9%, and 2.7%, respectively, and predicted amino acid sequence differences in env and S2 coding regions are 4.4% and 8.8%, respectively. Six conserved regions are characterized in Gp90. There     

马传染性贫血病毒驴白细胞弱毒疫苗株及其亲本强毒株前病毒核苷酸序列比较分析

以马传染性贫血病毒驴白细胞弱毒疫苗株(DLA-EIAV)感染细胞总DNA及其亲本强毒L株感染马外周血液白细胞中DNA为模板, 应用PCR方法分段扩增出DLA-EIAV和L株前病毒, 并将各段扩增产物克隆后进行测序. 根据国外发表的马传染性贫血病毒核苷酸序列, 推导出DLA-EIAV和L株全基因组核苷酸序列, 经比较分析, DLA-EIAV株前病毒基因组全长8266个碱基, EIAV L株前病毒基因组共有8235个碱基. DLA-EIAV株与其亲本L株和DA株核苷酸序列相比, 同源率分别为97.0%和 97.5%. DLA-EIAV株和L株相比, 长末端重复序列(LTR, 由U3, R, U5组成)、编码囊膜糖蛋白的基因env及ORF S2变异率很高, U3, R, U5, env及ORF S2核苷酸序列差异率分别达13.2%, 7.5%, 5.1%, 2.7%和3.9%, env基因及ORF S2推导的氨基酸序列差异率分别为4.4%和8.8%. 从EIAV高度变异的env各个毒株中鉴定出6个氨基酸序列保守区, 并发现中国的EIAV强、弱毒增强子区有转录因子GATA结合一致序列. 序列比较发现EIAV L比DLA株多2个N连接糖化位点. DLA-EIAV, L和DA株在ENH的主要差别是, DLA-EIAV株含有转录因子bHLH作用一致序列. 另外, DLA-EIAV株TAR茎的起始部位发生了改变, 形成一个尿嘧啶小泡. 这些变异对EIAV毒力的影响有待进一步研究.     

小球藻的甲基磺酸乙酯诱变及产EPA的条件研究

小球藻是海水养殖系统中常用的单细胞微藻,繁殖能力强,易于规模化培养,且可合成不饱和脂肪酸EPA、DHA等多种活性物质,在医疗和保健品开发中具有很高的应用价值。目前商业化培养的藻种多从自然界中直接获得,活性物质的产量较低且藻种易退化。为了获得EPA产量更高的藻种,该研究利用0.6%EMS对小球藻进行诱变,利用尼罗红染色法进行初筛并通过单细胞分离技术得到1株突变株EC1,通过气象色谱测定其EPA产量。结果表明:与出发藻株相比,突变株EPA(二十碳五烯酸)产量提高了8.97%。根据单因素试验确定突变株生长及产EPA的合适培养条件,再通过正交试验筛选出培养条件的优化组合,表明突变藻EC1株产EPA的较适条件为NaNO_375 mg·L~-1,p H7.5,昼夜温度17~15℃,接种量为12%,在此条件下培养7d其EPA的产量可达25.38 mg·g~-1,传代实验表明突变藻株具有较好的遗传稳定性。该研究结果为进一步利用小球藻规模化生产EPA奠定了基础。     

三角褐指藻的诱变育种及产EPA的条件研究

旨在获得不饱和脂肪酯EPA产量更高的藻种,利用0.6% EMS对三角褐指藻进行诱变。通过单细胞分离技术得到1株突变株EP1,其EPA产量比出发藻株提高了19.81%。结果显示,突变藻株产EPA的最适条件为:NaNO3 75 mg/L,pH7.5,昼夜温度17-15℃,接种量为12%。最适条件下培养7 d,其EPA产量可达26.77 mg/g。传代试验表明,突变藻株具有较好的遗传稳定性。