不同种类柑橘的密码子用法分析

采用高频密码子分析法，对甜橙Citrus sinensis、温州蜜柑C．unshiu、葡萄柚C．paradisi和柠檬C.limon等4种柑橘的蛋白质编码基因序列（eodon DNA sequence，CDS）进行了分析，计算出了柑橘同义密码子相对使用频率（relative frequency of synonymous codon，RFSC），确定出了4种柑橘的高频率密码子，发现不同种类柑橘偏爱密码子稍有差别．     

Codon optimization of the rabbit hemorrhagic disease virus (RHDV) capsid gene leads to increased gene expression in Spodoptera frugiperda 9 (Sf9) cells

Rabbit hemorrhagic disease (RHD) is contagious and highly lethal. Commercial vaccines against RHD are produced from the livers of experimentally infected rabbits. Although several groups have reported that recombinant subunit vaccines against rabbit hemorrhagic disease virus (RHDV) are promising, application of the vaccines has been restricted due to high production costs or low yield. In the present study, we performed codon optimization of the capsid gene to increase the number of preference codons and eliminate rare codons in Spodoptera frugiperda 9 (Sf9) cells. The capsid gene was then subcloned into the pFastBac plasmid, and the recombinant baculoviruses were identified with a plaque assay. As expected, expression of the optimized capsid protein was markedly increased in the Sf9 cells, and the recombinant capsid proteins self-assembled into virus-like particles (VLPs) that were released into the cell supernatant. Rabbits inoculated with the supernatant and the purified VLPs were protected against RHDV challenge. A rapid, specific antibody response against RHDV was detected by an ELISA in all of the experimental groups. In conclusion, this strategy of producing a recombinant subunit vaccine antigen can be used to develop a low-cost, insect cell-derived recombinant subunit vaccine against RHDV.     

Analysis of synonymous codon usage in chloroplast genome of Populus alba

The pattern of codon usage in the chloroplast genome of Populus alba was investigated. Correspondence analysis （a commonly used multivariate statistical approach） and method of effective number of codons （ENc）-plot were conducted to analyze synonymous codon usage. The results of correspondence analysis showed that the distribution of genes on the major axis was significantly correlated with the frequency of use of G＋C in synonymously variable third position of sense codon （GC3S）, （r=0.349）, and the positions of genes on the axis 2 and axis 3 were significantly correlated with CAI （r=-0.348, p〈0.01 and r=0.602, p〈0.01）. The ENc for most genes was similar to that for the expected ENc based on the GC3s, but several genes with low ENc values were lying below the expected curve. All of these data indicated that codon usage was dominated by a mutational bias in chloroplast gcnome ofP. alba. The selection in nature for translational efficiency only played a minor role in shaping codon usage in the chloroplast genome ofP alba.     

双软件多参数联合对草鱼同义密码子偏性的分析

密码子偏性分析数据有助于被用来构建高效表达载体,提高外源基因在宿主体内的表达效率。为了解草鱼密码子使用情况,以便在草鱼中高效表达外源目标蛋白来预防草鱼疾病的发生,应用Codon W及CAIcal软件分析了草鱼(Ctenopharyngodon idella)基因组中的208个蛋白质的编码序列。通过分别计算密码子中各个位置的 G C 含量、密码子适应指数（CAI）、有效密码子数（ENc）、密码子偏爱指数(CBI)、同义密码子的使用频率、最优密码子使用频率（FOP）等,分析了草鱼的密码子使用偏性,确定了草鱼的最优密码子。结果显示,草鱼密码子第 2位的G C 含量明显低于第 1、3 位,第3位表现出对以 G 或 C 碱基结尾的密码子偏向使用;所确定的23个最优密码子中,除终止密码子是以碱基A结尾外,有18个以碱基G或 C结尾,4个以碱基U结尾。所分析结果既可用于分析草鱼基因的组成结构,了解草鱼的分子进化状态,又可以通过所获的结果来指导对外源基因进行分子改造,提高外源基因在草鱼中的表达效率。     

双生病毒的同义密码子用法及其进化分析

 对双生病毒编码基因的变异进行了密码子用法分析,推测双生病毒基因的表达水平普遍不高,而外壳蛋白基因的表达水平相对较高,它们在密码子第3位上偏好使用A或T。双生病毒的密码子用法主要受到突变偏好和翻译选择等因素的影响。双生病毒的密码子使用具有基因特异性和一定程度的寄主特异性。基于相对同义密码子使用频率的聚类分析可以很好反映侵染单子叶植物和双子叶植物双生病毒之间的差异,也在一定程度上反映出双生病毒之间的亲缘关系。     

鸭肠炎病毒核衣壳蛋白密码子偏爱性分析

为给鸭肠炎病毒（DEV）核衣壳蛋白（NP）基因选择良好的宿主表达系统提供参考依据,本研究运用EM-BOSS软件包CHIPS和CUSP程序对DEVNP基因进行了密码子偏爱性分析。结果显示,DEV NP基因的ENC值为51.180,编码NP蛋白的A、I等氨基酸不同密码子的使用频率存在一定的差异;从与DEV NP基因密码子使用频率比值上看,大肠杆菌22个、酵母18个、鸭12个和人20个密码子存在较大的偏爱性。由此可见,编码DEVNP蛋白密码子出现频率较均一,且酵母等真核生物与其密码子偏爱性较为接近,可作为该基因体外表达宿主的首选。     

水牛瘦素受体基因密码子使用偏好性分析

试验选取27头河流型水牛和41头沼泽型水牛,以从NCBI数据库中下载的家牛、野牦牛、野牛、绵羊、山羊、小鼠和人的序列作对照,对编码瘦素受体（leptin receptor,LEPR）基因的密码子偏好性及水牛与其他物种间密码子使用的差异和进化关系进行了深入分析。结果发现,所有的密码子在河流型水牛和沼泽型水牛LEPR基因中均有使用,两种类型水牛偏好使用的密码子有28个,其中使用偏好性较强的密码子为AGA（RSCU≥2）,表明河流型水牛和沼泽型水牛LEPR基因密码子使用特征相似。水牛及参考物种LEPR基因均偏好使用以A/U结尾的密码子,但水牛与其他参考物种间偏好使用密码子的种类和数目有差异。密码子使用偏好聚类分析表明,河流型水牛与沼泽型水牛亲缘关系最近,先聚为一类,然后与家牛、野牦牛和野牛聚为一类,再与绵羊、山羊、小鼠和人等物种聚为一类。在密码子使用频率上,河流型水牛LEPR基因与酵母密码子偏好性差异小于与大肠杆菌和小鼠密码子偏好性差异,从而揭示酵母更适合作为水牛LEPR基因的外源表达系统。     

猪瘟病毒E2基因密码子偏爱性分析

本试验旨在阐明猪瘟病毒(classical swine fever virus,CSFV)囊膜结构(糖)蛋白E2基因的密码子使用规律,以期为选择高效的表达系统、探索基因功能提供理论依据.本研究运用EMBOSS在线分析软件的CHIPS和CUSP程序对CSFV E2基因进行密码子偏爱性分析.结果显示CSFV E2基因的CAI值为0.703,ENC值为53.161,表明E2在密码子使用上存在较明显的偏爱性,且第3位核苷酸尤其偏爱G或C;从与CSFV E2基因密码子使用频率比值上看,使用频率差异较大的在大肠杆菌有33个、酵母有25个、人有25个;CSFV E2基因共有34个稀有密码子,且还有多个稀有密码子多次串联成簇的情况出现.本试验结果表明酵母等真核生物与CSFV E2基因密码子偏爱性较为接近, 可作为其体外表达系统.     

Codon‐optimized expression of fish iridovirus capsid protein in yeast and its application as an oral vaccine candidate

Fish iridovirus causes systemic disease with high morbidity and mortality in various species of wild and farm‐raised fish, resulting in severe economic losses. Recently, frequent outbreaks of iridovirus infection have occurred among cultured fish in many Asian countries, emphasizing the need for a protective vaccine programme or the development of a suitable therapy. In this study, we expressed a recombinant major capsid protein (rMCP) of rock bream iridovirus (RBIV) from yeast using codon optimization. The rMCP in yeast was added to feed in an attempt to induce intestinal mucosal immunity for protection against and/or to reduce the severity of fish iridovirus infection. We found that fish immunized orally with rMCP underwent a successful induction of antibodies (P < 0.05) and were protected (P = 0.0001) against viral challenge. Based upon these results, oral administration of immunogenic protein as an antigen can be considered a useful method for implementation of vaccine programmes against iridovirus as well as other marine viral diseases.     

马铃薯卷叶病毒CP基因的突变及其原核表达

通过人工合成DNA的方法,对马铃薯卷叶病毒外壳蛋白(CP)基因第52～177核苷酸(126bp)这段序列进行了突变,将其中12个AGA、AGG、CGA等精氨酸稀有密码子变成了原核高效表达的同义密码子CGT与CGC,将另外两个AGA精氨酸密码子变成了错义密码。构建了突变基因的原核表达载体pBAD-LRCP2,Bsp1407Ⅰ与MssⅠ酶切及DNA测序结果表明,基因突变符合要求,表达载体的构建正确。在37℃,大肠杆菌工程株TOP10(pBAD-LRCP2)用0.2%L-阿拉伯糖诱导培养4h,SDS-PAGE显示蛋白图谱上有一条36ku的诱导表达的融合蛋白条带,结果表明突变基因在PBAD启动子驱动下在大肠杆菌中实现了表达。