石岐杂鸡r-干扰素基因的克隆与序列分析研究

华南农业大学动物科学学院吕英姿、毕英佐和曹永长三位先生对广东石歧杂鸡r-干扰素基因的克隆与序列作了分析,他们根据现有鸡r-干扰素基因序列设计引物,应用反转录-聚合酶链反应（RT-PCR）技术,从ConA诱导培养的鸡脾淋巴细胞中扩增得到石歧杂鸡r-干扰素（SqzChIFN-r）基因,将其克隆到PGEM-T载体中,进行序列测定,结果表明,经克隆得到的那种ChIFN—r基因的开放阅读框由492个核苷酸组成,它与其他ChIFN—r基因的同源性达到99％,与火鸡r-干扰素基因的同源性为96％,与鸭r-干扰素基因的同源性为81％。由此,他们推导石歧杂鸡IFN-氨基酸序列与其他已知的ChIFN—r氨基酸序列完全相近或一致,同时与火鸡和鸭IFN—r氨基酸同源性分别为97％和67％。此说明石岐杂鸡r-干扰素基因的同源性与鸡类的较近,与其他不同种类的则较远。     

鸡IL-18成熟蛋白基因的克隆及分子进化分析

根据已发表的鸡白介素18(IL-18)基因序列设计合成引物,以植物凝集素(PHA)和脂多糖(LPS)激活的AA肉鸡脾细胞mRNA为模板,通过RT-PCR扩增出编码鸡IL-18成熟蛋白的eDNA。将该eDNA克隆于pUCm-T载体,并对其进行测序,结果表明所克隆的核苷酸片段包含了全部成熟蛋白编码基因,成熟蛋白编码区507个核苷酸,编码169个氧基酸。把该基因编码的鸡成熟IL-18蛋白氨基酸序列与已公布的禽及哺乳动物IL-8成熟蛋白基因氧基酸序列进行比较,其同源性分别在96．5％～100％和20．1％～26．6％之间,分子系统进化树分析表明鸡IL-18与哺乳动物IL-18有共同的祖先,亲源关系较近,但在免疫系统选择性压力下,形成独特的种族特异性。鸡IL-18基因的克隆为体外表达鸡IL-18蛋白及作为免疫佐剂应用于预防接种的研究奠定了基础。     

鸡二价金属转运蛋白1 cDNA的克隆与序列分析

鸡二价金属转运蛋白1（divalent metal transporter 1, DMT1）在动物胃肠道锰吸收过程中起重要作用.根据哺乳动物Dmt1同源蛋白氨基酸序列的保守性设计引物,应用3′RACE(rapid amplification of cDNA ends)技术,扩增并克隆获得鸡小肠Dmt1 cDNA 3′端1 289bp和1 092bp的2种片段,发现其3′端翻译区和非翻译区存在差异. 根据鸡Dmt1 cDNA 3′端片段的测序结果设计引物,扩增获得1个与3′端片段部分重叠的鸡Dmt1 cDNA 5′端907 bp片段,并对其进行了克隆测序. 根据鸡小肠Dmt1 3′RACE片段和5′RACE片段序列信息进行拼接,从而获得鸡小肠Dmt1 cDNA全序列信息.结果表明,鸡小肠Dmt1 cDNA有2种形式,1种全长为1 972个核苷酸,其中5′非翻译区为104个核苷酸,编码区1 695个核苷酸,3′非翻译区为173个核苷酸,编码1个含564个氨基酸残基的蛋白质;另1种形式为1 775个核苷酸,其中5′非翻译区为104个核苷酸,编码区1 593个核苷酸,3′非翻译区为78个核苷酸,编码1个含530个氨基酸残基的蛋白质.据鸡Dmt1 cDNA推测出的2种形式蛋白质的氨基酸序列与人、大鼠和小鼠的Dmt1蛋白具有高度同源性,它们的同源性分别为82%、82%、80%,和 84%、84%、83%. 对推测氨基酸序列进行疏水性和跨膜区分析表明,Dmt1蛋白为1种跨膜整合蛋白,具有膜转运蛋白糖基化位点和底物结合位点的保守序列.     

鸡传染性支气管炎中国地方分离毒株LX4mRNA1ORF1的遗传变异特征

应用RT-PCR方法扩增了冠状病毒鸡传染性支气管炎中国分离毒株LX4的mRNA1 ORF1,并将其进行了克隆、序列测定和分析.表明LX4mRNA1基因包含2个ORF,其中ORF1a由11 916个核苷酸组成,编码一条3 971个氨基酸残基组成的多肽.ORF1b由7 950个核苷酸组成,编码2 649个氨基酸残基组成的多肽.与美国Beaudette株对应的ORF1a核苷酸及推导的氨基酸序列同源性均为85%,与对应的ORF1b的同源性分别为89%和95%.二者ORF1a和ORF1b重叠区推测的"滑脱序列"(slippery sequence)核苷酸序列一致,"假结"(pseudoknot)基本结构相似.不同之处在于"假结"第2环中LX4的第7位为A,而Beaudette株在该位置为G.与Beaudette株比较,LX4 ORF1a核苷酸变异最频繁的区域集中在第2 656～3 098位碱基处,且在该区域有60个碱基的插入,导致推导的氨基酸序列插入了20个氨基酸残基.LX4 ORF1b序列缺失9个核苷酸,主要集中在第5 168～5 181位之间,导致对应的氨基酸缺失3个.但这些差异是否与病毒的生物学特性有关不明.     

香鱼巨噬细胞炎性蛋白-2基因的克隆、序列分析及表达

巨噬细胞炎性蛋白-2(MIP-2)是一种重要的趋化因子,可趋化中性粒细胞到炎症部位,从而消除炎症反应。从香鱼巨噬细胞转录组测序中获得MIP-2基因,阅读框序列长为318个核苷酸,编码一个由105个氨基酸组成、相对分子质量为11.6kD的前体蛋白。N端19个氨基酸为信号肽序列。氨基酸序列分析表明,香鱼MIP-2与白斑狗鱼MIP-2的氨基酸同源性最高,为54%。健康香鱼MIP-2基因mRNA主要在脾、肾、脑、鳃中表达,在肝、心、肌肉、肠中表达量次之。实时荧光定量PCR结果显示,鳗利斯顿氏菌侵染香鱼后,各组织中MIP-2基因mRNA表达量均呈上调趋势。尤其以肾组织和肌肉组织中变化最显著。以上结果表明,香鱼MIP-2基因表达与鳗利斯顿氏菌的侵染密切相关,揭示了MIP-2可能在香鱼抗菌免疫反应中具有重要的作用。     

北京鸭卵磷脂胆固醇酰基转移酶的cDNA和蛋白质的结构分析

为获得不易感动脉粥样硬化动物北京鸭卵磷脂胆固醇酰基转移酶 (LCAT)的cDNA和蛋白质序列 ,分析其结构特点 .以从北京鸭肝脏mRNA反转录获得的cDNA一链为模板 ,应用SMART RACE技术 ,获得了北京鸭LCAT的cDNA序列 ,推导出其蛋白质氨基酸序列 ,应用分子生物学软件对该蛋白的一级、二级结构进行分析和比较 .北京鸭LCATcDNA (在GenBank中的注册号为AF32 4 887)全长 195 3bp ,其中开放阅读框架 135 6bp ,编码 4 5 1个氨基酸 ,包括一个由 2 3个氨基酸构成的疏水性信号肽和一个由 4 2 8个氨基酸组成的成熟蛋白 .该成熟蛋白比人LCAT在C端多 12个氨基酸 ,其与鸡、人、家兔的同源性依次为 98%、83%和 82 % .与其它种属LCAT蛋白序列的比较结果表明 ,北京鸭LCAT蛋白质序列虽然在长度上和结构上与其它种属有一定的差异 ,但序列中与酶催化活性相关的序列均非常保守     

犬冠状病毒流行株膜蛋白基因序列分析及其表达研究

对国内分离的犬冠状病毒(CCoV)DXMV、V1和V2流行毒株膜蛋白(M)基因进行了扩增、测序和遗传进化分析.3个CCoV流行毒株M基因全长均为792bp,编码263个氨基酸,其中前17个氨基酸为信号肽.DXMV、V1和V2流行株与Insavc-1疫苗株M基因相比,核苷酸的同源性分别为92.6% 、90.9%和91.6%,推导的氨基酸序列的同源性分别为92.5%、92.0%和92.3% ,在M基因前1/3区域内存在变异,其中74-76、120-124和131-135三个区域变异较大.国内DXMV、V1、V2、NJ1和NJ1-17 5个流行株M基因核苷酸同源性为96.6%,推导的氨基酸序列同源性为96.4%,显示出很高的保守性.基于M基因的遗传进化分析表明,目前国内绝大多数CCoV流行毒株都属于CCoV基因II型,只有Fox3-1和Rac2-1两个毒株属于基因I型.另外,将DXMV株M基因亚克隆到pET28a中,在BL21(DE3)中实现了M蛋白的表达,表达量约占菌体蛋白的10.2%.     

一步法扩增克隆IBDV上海超强毒VP2-4-3基因

分离、纯化了鸡传染性法氏囊病病毒超强毒 (vvIBDV) 上海株SH95的病毒核酸dsRNA,应用随机引物将RNA反转录成cDNA,以此为模板一步扩增出A片段前体融合蛋白基因即VP2-4-3基因,将其克隆入pGEM-T载体,并进行序列分析,其与超强毒株HK46的核苷酸序列的同源性达98%,整个基因有5个氨基酸差异,同源性达99.51%(1007/1012).     

传染性法氏囊病病毒不同分离株vp2基因部分核酸序列分析

根据已报告的传染性法氏囊病病毒(Infectious bursal disease virus,IBDV)cDNA序列,设计引物,用RT-PCR扩增CH(鸡)、DU(鸭)、GE(鹅)和SP(麻雀)四种不同源IBDV分离株的vp2基因高变区.核酸序列测定分析表明,四种不同源IBDV分离株vp2基因高变区的同源性为97%,推导编码蛋白氨基酸序列的同源性98%,两个亲水区和七肽区的氨基酸序列完全一致.本研究结果提示,自然感染IBDV的鸭、鹅和麻雀不仅可成为病毒携带者或传染源,而且在病毒变异中起一定作用.     

禾谷镰孢菌α-微管蛋白基因克隆及其与多菌灵抗药性关系分析

根据禾谷镰孢菌参考菌株NRRL310 84 (PH 1)的α- 微管蛋白基因核苷酸序列设计 4对引物 ,采用PCR方法克隆并测序了禾谷镰孢菌 (Fusariumgraminearum)对多菌灵 (MBC)不同敏感性表型的 6个中国菌株的α 微管蛋白基因全序列。DNA序列对照表明中国的 3个敏感菌株和 3个抗药菌株的α- 微管蛋白基因核苷酸序列同源性没有差异 ,多菌灵抗药性与α- 微管蛋白无关。该基因全长 1718bp ,含有 6个内元 ,编码 4 4 9aa ;与NRRL310 84的α- 微管蛋白基因核苷酸序列同源性为 99% ,存在 5个差异核苷酸 ,与其所编码的氨基酸序列同源性为 99 78% ;与其他 6种真菌α- 微管蛋白基因所编码的氨基酸序列同源性为 37%～ 86 %。     

cDNA cloning and functional analysis of goose interleukin-2

cDNA encoding goose IL-2 (GoIL-2) was cloned from Con A-stimulated goose splenic mononuclear cells (SMC) using oligonucleotide primers based on the conserved sequence of duck (DuIL-2), chicken (ChIL-2) and turkey IL-2s (TuIL-2). The GoIL-2 cDNA is 718nt long, which contains an open reading frame (ORF) of 423 base pairs encoding a protein of 141 aa. The GoIL-2 shows, respectively, 79%, 82-85%, and 91-92% identities with TuIL-2, ChIL-2 and DuIL-2 in cDNA, and also shows, respectively, 63%, 63-64%, and 82-85% identities with TuIL-2, ChIL-2 and DuIL-2 in amino acid sequence. Recombinant GoIL-2 (rGoIL-2) protein expressed in Escherichia coli has an approximate molecular weight of 18kDa. The rGoIL-2 has biological effect on goose and duck as well as chicken lymphocytes in a dose-dependent manner, though the effect on duck and chicken lymphocytes has been found to be relatively weak. In addition, rGoIL-2 also strengthens goose immune responses induced by vaccinating the inactivated oil emulsion vaccine against avian influenza virus. The monoclonal antibodies (mAb) to rGoIL-2 recognized the binding epitopes of nature GoIL-2 protein expressed in vero cells. Antiserum and mAb 5B10 to rGoIL-2 can inhibit the biological activity of rGoIL-2 and endogenous GoIL-2. The results, at the first time, indicated that goose IL-2 reserves species-specialties in the biological functions and can be used as a potential immunoadjuvant for goose vaccination and immunotherapeutic purposes. Finally, the mAbs to rGoIL-2 also provide a useful tool for further immunobiological studies of IL-2 in avian immune systems.     

cDNA cloning, expression, and characterization of an arylphorin-like hexameric storage protein, AgeHex2, from the mulberry longicorn beetle, Apriona germari

An arylphorin-like hexameric storage protein, AgeHex2, cDNA was cloned from the mulberry longicorn beetle, Apriona germari (Coleoptera, Cerambycidae), larval cDNA library. The complete cDNA sequence of AgeHex2 is comprised of 2,088 bp encoding 696 amino acid residues. The AgeHex2 had four potential N-glycosylation sites. The AgeHex2 contained the highly conserved two larval storage protein signature motifs. The deduced protein sequence of AgeHex2 showed high homology with A. germari hexamerin1 (51% amino acid identity), Tenebrio molitor hexamerin2 (49% amino acid identity), T. molitor early-staged encapsulation inducing protein (43% amino acid identity), and Leptinotarsa decemlineata diapause protein1 (43% amino acid identity). Phylogenetic analysis further confirmed the AgeHex2 is more closely related to coleopteran hexamerins than to the other insect storage proteins. Northern blot analysis confirmed that the AgeHex2 showed fat body-specific expression. The cDNA encoding AgeHex2 was expressed as a 75-kDa protein in the baculovirus-infected insect cells. Furthermore, N-glycosylation of the recombinant AgeHex2 was revealed by tunicamycin to the recombinant virus-infected Sf9 cells, demonstrating that the AgeHex2 is N-glycosylated. Western blot analysis using the polyclonal antiserum against recombinant AgeHex2 indicated that the AgeHex2 corresponds to a 75-kDa storage protein present in the A. germari larval hemolymph.     

中华蜜蜂磷酸甘油变位酶（PhosphoglycerateMutase）基因的序列分析

在中华蜜蜂(Apis cerana)工蜂毒腺cDNA库内发现了一个插有1104bp外源片段的克隆,内含一个765bp的开放阅读框架(ORF),编码一个含有254．个氨基酸残基的依赖于2,3一二磷酸甘油酸的磷酸甘油变位酶(dPGAM),催化3一磷酸甘油和2一磷酸甘油之间的转化。推测的氨基酸序列与其他7种生物的dPGAM的相似性很高(39％-88％),而与其他4种不依赖于2,3-二磷酸甘油酸的磷酸甘油变位酶(iPGAM)的相似性则很低(10％-12％),氨基酸序列的多重联配表明组成dPGAM活性位点的氨基酸残基在包括中华蜜蜂在内的所有生物体内是十分保守的,Ac—PGAM是一种典型的dPGAM。这是昆虫纲中继在果蝇中发现PGAM基因后的第2个昆虫dPGAM基因,其对PGAM基因的结构与功能研究及对昆虫的分子生物学研究具有意义。同时,对PGAM的进化关系的分析表明该基因可以用作研究物种系统关系的一个依据。     

林麝干扰素α基因克隆、表达及转录调控分析

干扰素在病毒性疾病的防治上,具有很高的临床应用前景.为了林麝干扰素的研究与应用,使用同源克隆方法首次克隆得到9条林麝干扰素α基因序列,序列全长均为570 bp,编码189个氨基酸,前23个氨基酸为信号肽,具有4个保守的半胱氨酸残基和5个保守的脯氨酸残基.9种亚型之间,核酸序列同源性为97.0％-99.6％,氨基酸序列同...     

The nucleotide sequences of the ponA and ponB genes encoding penicillin-binding protein 1A and 1B of Escherichia coli K12

Penicillin-binding proteins 1A and 1B of Escherichia coli are the major peptidoglycan transglycosylase-transpeptidases that catalyse the polymerisation and insertion of peptidoglycan precursors into the bacterial cell wall during cell elongation. The nucleotide sequence of a 2764-base-pair fragment of DNA that contained the ponA gene, encoding penicillin-binding protein 1A, was determined. The sequence predicted that penicillin-binding protein 1A had a relative molecular mass of 93 500 (850 amino acids). The amino-terminus of the protein had the features of a signal peptide but it is not known if this peptide is removed during insertion of the protein into the cytoplasmic membrane. The nucleotide sequence of a 2758-base-pair fragment of DNA that contained the ponB gene, encoding penicillin-binding protein 1B, was also determined. Penicillin-binding protein 1B consists of two major components which were shown to result from the use of alternative sites for the initiation of translation. The large and small forms of penicillin-binding protein 1B were predicted to have relative molecular masses of 94 100 and 88 800 (844 and 799 amino acids). The amino acid sequences of penicillin-binding proteins 1A and 1B could be aligned if two large gaps were introduced into the latter sequence and the two proteins then showed about 30% identity. The amino acid sequences of the proteins showed no extensive similarity to the sequences of penicillin-binding proteins 3 or 5, or to the class A or class C beta-lactamases. Two short regions of amino acid similarity were, however, found between penicillin-binding proteins 1A and 1B and the other penicillin-binding proteins and beta-lactamases. One of these included the predicted active-site serine residue which was located towards the middle of the sequences of penicillin-binding proteins 1A, 1B and 3, within the conserved sequence Gly-Ser-Xaa-Xaa-Lys-Pro. The other region was 19-40 residues to the amino-terminal side of the active-site serine and may be part of a conserved penicillin-binding site in these proteins.     

Cloning and characterization of chicken IL-10 and its role in the immune response to Eimeria maxima

We isolated the full-length chicken IL-10 (chIL-10) cDNA from an expressed sequence tag library derived from RNA from cecal tonsils of Eimeria tenella-infected chickens. It encodes a 178-aa polypeptide, with a predicted 162-aa mature peptide. Chicken IL-10 has 45 and 42% aa identity with human and murine IL-10, respectively. The structures of the chIL-10 gene and its promoter were determined by direct sequencing of a bacterial artificial chromosome containing chIL-10. The chIL-10 gene structure is similar to (five exons, four introns), but more compact than, that of its mammalian orthologues. The promoter is more similar to that of Fugu IL-10 than human IL-10. Chicken IL-10 mRNA expression was identified mainly in the bursa of Fabricius and cecal tonsils, with low levels of expression also seen in thymus, liver, and lung. Expression was also detected in PHA-activated thymocytes and LPS-stimulated monocyte-derived macrophages, with high expression in an LPS-stimulated macrophage cell line. Recombinant chIL-10 was produced and bioactivity demonstrated through IL-10-induced inhibition of IFN-gamma synthesis by mitogen-activated lymphocytes. We measured the expression of mRNA for chIL-10 and other signature cytokines in gut and spleen of resistant (line C.B12) and susceptible (line 15I) chickens during the course of an E. maxima infection. Susceptible chickens showed higher levels of chIL-10 mRNA expression in the spleen, both constitutively and after infection, and in the small intestine after infection than did resistant chickens. These data indicate a potential role for chIL-10 in changing the Th bias during infection with an intracellular protozoan, thereby contributing to susceptibility of line 15I chickens.     

Molecular cloning and sequencing of the banded dogfish (Triakis scyllia) interleukin-8 cDNA

The dogfish (Triakis scyllia) interleukin-8 (IL-8) cDNA was isolated from mitogen-stimulated peripheral white blood cells (WBCs) utilising the polymerase chain reaction (PCR). The cDNA sequence showed that the dogfish IL-8 clones contained an open reading frame encoding 101 amino acids. A short 5' untranslated region (UTR) of 70 nucleotides and a long 3' UTR of 893 nucleotides were also present in this 1.2-kb cDNA. Furthermore, the 3' UTR of the mRNA contained the AUUUA sequence that has been implicated in shortening of the half-life of several cytokines and growth factors. The predicted IL-8 peptide had one potential N-linked glycosylation site (Asn-72-Thr-74) that is not conserved in other vertebrates. It also contained four cysteine residues (Cys-34, 36, 61 and 77), which are characteristic of CXC subfamily cytokines and found in all vertebrates, to date. The dogfish IL-8 lacked an ELR motif as found in the lamprey and trout. Comparison of the deduced amino acids showed that the dogfish IL-8 sequence shared 50.5, 41.2, 37.1 and 40.4-45.5% identity with the chicken, lamprey, trout and mammalian IL-8 sequences, respectively.     

Mutational analysis of chicken interleukin 2

Chicken interleukin 2 (chIL-2) has low, but significant, homology to both mammalian IL-2 and mammalian IL-15. In view of its unique phylogenetic position and potential use as a vaccine adjuvant, a detailed mutational analysis for critical functional sites was undertaken. It was found that Asp17 is a critical N terminal contact site for binding to the putative chIL-2 receptor, which is similar to results obtained for mammalian IL-2 and IL-15. Analysis of the C terminus did not reveal a single critical amino acid. However, deletion mutant studies demonstrated that removal of C terminal amino acids yielded proteins with decreased bioactivity and that this decrease was a function of the number and kind of amino acids removed. This study is the first non-mammalian IL-2 mutational analysis and proposes a model for the interaction between chIL-2 and its receptor.