【作者】 何睿林；

【导师】 梁宁生；

【作者基本信息】 广西医科大学 ， 生化药理， 2011， 博士

【摘要】 目的：分析亲缘关系密切,但又分属不同种类的灵长类生物ⅡA型磷脂酶A2基因的相关情况,了解ⅡA型磷脂酶A2分子进化的一些特点。方法：收集已知的灵长类生物人(Homo sapiens)、猩猩(Pan troglodytes)和猴(Macaca mulatta)的ⅠB型和ⅡA型磷脂酶A2 (phospholipase k2,PLA2)的基因和mRNA顺序资料,对两型PLA2进行碱基替代的相关分析,包括转换和颠换、颠换／转换比；碱基替代发生在编码区和非编码区的情况；以及替代发生在密码子Ⅰ、Ⅱ、Ⅲ位上的情况等。结果：灵长生物PLA2编码区基因发生碱基替代时,ⅠB型和ⅡA型PLA2碱基总替代数在发生人和猩猩之间最少,而人和猴、猴和猩猩之间则碱基替代明显增多,约为前者的5倍和3倍多；ⅠB型PLA2碱基替代在人和猩猩之间以密码子Ⅰ、Ⅲ位最多,各占40%,第Ⅱ位者最少占20%；而在人和猴、猴和猩猩之间按替代频率高低依次为Ⅲ>Ⅱ>Ⅰ位,发生于第Ⅰ、Ⅱ位的总碱基替代百分率为48%-50%,低于发生于第Ⅲ位的50%-52%；人和猴、猴和猩猩之间碱基转换和颠换的比值>1,而人和猩猩之间比值<1。与ⅠB型PLA2相比,ⅡA型PLA2在三种灵长生物之间的碱基总替代数更多(62 vs 54),碱基替代在密码子中所占的比例也更高；人、猴和猩猩三者之间的碱基替代均以密码子第Ⅲ位最多,占40.7%-50%,发生于Ⅰ、Ⅱ位者在25.9%-33.3%之间,但Ⅰ、Ⅱ位两个位点总的碱基替代率达55.5%-59.2%,明显高于发生于第Ⅲ位者；突变的性质方面,人和猩猩、猴和猩猩之间碱基转换多于颠换,而人和猴之间则相反,颠换略多于转换。在非编码区5’端在人和猩猩间ⅠB型PLA2没有碱基替代,而工ⅡA型PLA2则有2处转换,且人和猴、猴和猩猩之间总替代数达60多次,其中颠换明显多于转换,人和猴、猩猩三者之间的碱基总替代以及转换和颠换占5’端碱基数的百分比明显高于ⅠB型。在非编码区3’端,ⅠB型PLA2在三种灵长生物之间均有1次颠换,人和猩猩之间无碱基转换,而人和猴、猴和猩猩之间各有4次和5次转换,且碱基转换无论数目还是比例都多于颠换。ⅡA型PLA2在三种灵长生物之间的碱基总替代数高于ⅠB型,并且人和猩猩、人和猴之间总替代数及颠换数占3’端碱基数的百分比也明显高于ⅠB型。结论：1.ⅡA型PLA2分子具有较快的进化速度,存在加速进化现象；2.ⅡA型PLA：加速进化可能与协同进化有关。目的：研究中国人不同个体间ⅡA型磷脂酶A2(phospholipase A2, PLA2) cDNA和氨基酸顺序的情况,并与已知外国人种的ⅡA型PLA2进行比较,了解人ⅡA型PLA2分子的进化特点。方法：12名因急性或慢性阑尾炎急性发作施行阑尾切除手术的患者,取其手术切除的阑尾炎症组织50-100mg,以Trizol试剂法匀浆提取总RNA,利用RT-PCR方法分别扩增其ⅡA型PLA2 cDNA,经电泳确认条带清晰的PCR产物送专业测序公司行DNA双向测序,分析测序所获得的DNA及由其推导的氨基酸顺序,并与已知外国人种ⅡA型PLA2的进行比较,观察碱基和氨基酸取代情况,结合测序图谱,分析其分子进化的特点。结果：从12名不同中国人个体的阑尾炎症组织顺利扩增得到ⅡA型PLA2cDNA,并推导出相应的氨基酸顺序,与已知外国人种的相比较,发现：①扩增得到的ⅡA型PLA2 cDNA顺序与已知外国人种的基本一致；但存在单核苷酸多态性(Single Nucleotide polymorphism, SNPs)。对测序图谱的分析发现,在12例样本中有2例可见突变的单等位基因SNPs位点,分别位于密码子32位和44位,且都发生在突变热点CpG二核苷酸位点上,并存在杂合(CG＆CT)和纯合现象。②由ⅡA型PLA2 cDNA推导的氨基酸顺序与已知外国人种的完全一致。结论：①中国人不同个体间ⅡA型磷脂酶A2的cDNA顺序高度保留,与已知其他人种也一样。多态性位点发生在密码子32和44位上,且都发生在突变热点CpG二核苷酸位点上；②中国人种ⅡA型磷脂酶A2分子一级结构与其他人种完全一致,说明这一在宿主天然免疫系统中发挥重要作用的分子在不同人种间高度保留。目的：了解人ⅡA型磷脂酶A2 cDNA片段和婴儿利什曼原虫DNA相似性的情况和原因。方法：搜索NCBI网站基因库并获取人ⅡA型磷脂酶cDNA资料,然后利用BLAST软件,以人ⅡA型磷脂酶cDNA顺序中的片段以每相邻20个碱基(bp)为一组(如1-20；10-30；20-40…,以此类推直至结尾),作为查询序列与核酸数据库进行逐一比对,将获得的一致性片段进行收集、整理并分析、汇总利什曼原虫DNA的相关信息。结果：发现当DNA片段长度≥17bp,人ⅡA型磷脂酶A2 cDNA与婴儿利什曼原虫DNA之间仅有4个片段一致；而在片段长度≥16bp时,则两者之间有9个片段完全相同,并且这些序列集中分布于婴儿利什曼原虫36条染色体的29、30号染色体上。结论：人ⅡA型磷脂酶cDNA片段与婴儿利什曼原虫DNA片段之间具有一致性,可能与基因由原虫向人的横向传递有关。目的：了解灵长生物的抗菌蛋白ⅡA型磷脂酶A2氨基酸取代的特点及其与分子进化的关系。方法：对人、猩猩和猴3种哺乳类灵长生物的ⅡA型磷脂酶A2的氨基酸顺序进行两两比较,并与ⅠB型磷脂酶A2的相对照,分析它们之间相互取代的位点、频率和性质(极性、非极性；酸性、中性、碱性),利用软件Cn3D4.1构建其蛋白分子的三维晶体结构,了解灵长类(人、猩猩、猴)ⅡA型磷脂酶A2晶体结构中发生氨基酸取代(碱基非同义取代)的位置和区域,总结出它们的变化规律,并与杀菌功能相联系。结果：(1)人和猩猩、人和猴、猩猩和猴之间ⅡA型磷脂酶A2蛋白分子分别有4、12、12个氨基酸残基发生取代,且主要发生在带电荷的氨基酸；而在发生的氨基酸取代中,又以带正电荷的氨基酸(碱性氨基酸)为主；(2)氨基酸取代主要集中在有限的4个区域,且为功能重要的区域。结论：哺乳类灵长生物ⅡA型磷脂酶A2的蛋白质分子进化较快,这可能是它能够具有杀菌活性的原因。目的：观察衍生自人ⅡA型磷脂酶A2 (phospholipase A2 ,ⅡA型PLA2)碳末端(C-terminal)26个氨基酸残基的多肽对不同细菌在体外的杀菌效应,并探讨其杀菌作用机制。方法：根据人ⅡA型PLA2C末端26个氨基酸残基的顺序,合成为肽P26。采用琼脂铺板计数法,将不同浓度的多肽分别与6种细菌(金黄色葡萄球菌、炭疽杆菌、枯草杆菌、大肠杆菌、变形杆菌和绿脓杆菌)在37℃孵育2h,然后铺板并置于37℃恒温箱培养18-24h,记录每一琼脂板上的菌落生成数(CFU),计算出多肽作用后对细菌的杀灭百分率。结果：肽P26对金黄色葡萄球菌、枯草杆菌、炭疽杆菌等革兰阳性(G+)菌的杀菌作用较强；对大肠杆菌、变形杆菌和绿脓杆菌等革兰阴性(G-)菌的杀菌作用较弱。结论：衍生自人ⅡA型PLA2C末端26个氨基酸残基的多肽有杀菌作用,推测其具有和ⅡA型PLA2及其他抗菌多肽相似的杀菌机制。目的：研究人ⅡA型磷脂酶A2 (phospholipase A2,ⅡA型PLA2)C末端衍生多肽P26经修饰和改造后对不同细菌的杀菌效应,分析其作用差异及机制。方法：以人ⅡA型PLA2氨基酸顺序C末端26个氨基酸残基为模板,合成线性多肽P26,然后对此线性多肽分别进行修饰和改造,一是将其环化得到环状多肽,二是将其中的4个碱性氨基酸残基分别替换为非极性疏水性氨基酸和极性中性氨基酸得到一条新的重组多肽。采用琼脂铺板计数法,将不同浓度的三种多肽分别与6种细菌(G+菌的枯草杆菌、炭疽杆菌和金黄色葡萄球菌；G-菌的大肠杆菌、变形杆菌和绿脓杆菌)在37℃孵育2h,然后铺板并置于37℃恒温箱培养18-24h,记录每一琼脂板上的菌落数(CFU),并计算多肽作用后的杀菌率。结果：直链肽和环肽P26对G+菌(包括金黄色葡萄球菌、枯草杆菌、炭疽杆菌)有较强的杀菌活性,但环肽的作用更强；对G-菌(包括大肠杆菌、变形杆菌和绿脓杆菌)的杀菌作用较弱,而环肽的作用更弱；改造后的重组肽P26对G+菌的杀菌活性明显下降,对G-菌则几乎不起作用。结论：衍生自人ⅡA型PLA2C末端的直链肽和环肽P26对G+菌有较强的杀菌活性,环肽作用更强,推测与其环状结构形成有关；但将其中的4个碱性氨基酸残基替换改造为非碱性氨基酸后的重组肽P26对G+菌的杀菌活性明显下降。更多还原

【Abstract】 Objective:To analyse the information about the gene of the group IIA phospholipase A2 (PLA2) on several primates which have close genetic relationship but dividing into distinc species, to get the message on characteristics of molecular evolution about group IIA phospholipase A2.Methods:All known mRNA and genes data of group IIA PLA2 on primates, which including of Homo sapiens, Pan troglodytes and Macaca mulatta, were collected and compared with the data of group I B PLA2. To analyze base substitutions of transition and transversion, ratio of transition and transversion; and the change of base substitutions took place in coding region and non-coding region, position of the site of the triplet codon, etc.Results:When base replacement occurred in the gene coding region of Primate Phospholipase A2, the total number of base replacement of type IIA and type I B PLA2 was the minimum between Homo sapiens and Pan troglodytes. However, the base replacement increased significantly between Homo sapiens and Macaca mulatta, as well as between Macaca mulatta and Pan troglodytes, and it was about five times and three times to the former. The base replacement observed in site I and site III of type I B PLA2 codon between Homo sapiens and Pan troglodytes was at the most, each with fourty percentage, and it was twenty percentage at least on site II on codon; For Homo sapiens and Macaca mulatta, Macaca mulatta and Pan troglodytes, according to the bases replacing frequency from high to low, it wasⅢ>Ⅱ>Ⅰsite of codon in turn, the percentage of total base substitution occurred in siteⅠandⅡwas fourty-eight percent to fifty percent, lower than that occurred in the siteⅢfrom fifty percent to fifty-two percent. And the ratio of the base transition and transversion between Homo sapiens and Macaca mulatta, Macaca mulatta and Pan troglodytes, was more than one, but lower than one between Homo sapiens and Pan troglodytes. Compared with typeⅠB PLA2, the total number of base replacement of type IIA PLA2 among the three species of Primate biology was more (62 vs 54), and the proportion of bases replacement of codons is also higher; the base replacement was observed at most on siteⅢof codon for type IIA PLA2 among Homo sapiens, Macaca mulatta and Pan troglodytes, accounting from 40.7 percent to 50 percent, it was occurred in the siteⅠand siteⅡof codon from 25.9 percent to 33.3 percent, but the rate of total base replacement was from 55.5 percent to 59.2 percent for the two sites ofⅠandⅡof codon, it was higher significantly than the siteⅢof codon. For the characteristic of mutations, the bases transition was more than bases transversion between Homo sapiens and Pan troglodytes, as well as between Macaca mulattas and Pan troglodytes. In contrast, bases transversion was more than transition slightly between Homo sapiens and Macaca mulatta.Conclusions:The rate of evolution of group IIA PLA2 was moderate, and acclerated evolution occurs in the group IIA PLA2 gene on primates. It may be associated with concerted evolution. OBJECTIVE:To investigate the properties of cDNA and amino acid sequence of human group IIA secreted phospholipase A2 (phospholipase A2, PLA2) on different individuals of Chinese, and compared to those of the known foreign species, understanding the features on molecular evolution of human group IIA secreted PLA2.METHODS:12 patients due to acute appendicitis or chronic appendicitis with acute exacerbation of the purposes of appendectomy, whichever was the surgical removal of appendicitis inflammation 50-100mg, the total RNA extracted by Trizol reagent, Reverse Transcription Polymerase Chain Reaction (RT-PCR) were used to amplify the cDNA of group IIA secreted PLA2, PCR product bands confirmed by agarose gel electrophoresis, and then sent the PCR product with clear electrophoretic bands to a professional sequencing company for forward and reverse DNA sequencing, analyzed DNA sequence obtained and its deduced amino acid sequence, and compared them with those of the known alien species, observed the base and amino acid replacement, and combined with DNA sequencing profiles, analyzed the characteristics of their molecular evolution.RESULTS:The cDNA of group II A secreted PLA2 amplified successfully by appendicitis symptoms organizations from 12 different individuals of Chinese, and deduced the corresponding amino acid sequence, when they were compared with the known alien species, It was found that:The amplified cDNA sequence of groupⅡA secreted PLA2 were basically the same as the known alien species with the exception of single nucleotide polymorphisms (Single Nucleotide polymorphism, SNPs). Analysis of the sequence Map, samples in 12 cases,2 cases of single mutant alleles of SNPs visible sites are located in codon 32 and codon 44, and mutations occured in hot spot locus of CpG dinucleotide with heterozygous (CG & CT) and homozygous phenomenon. The amino acid sequence deduced from group IIA secreted PLA2 cDNA has exactly the same structure as those of the foreign ethnic groups.CONCLUSIONS:The cDNA sequence of human group II A secreted phospholipase A2 in different individuals of Chinese was highly conserved, as well as those of other ethnic groups, polymorphism was observed in codon 32 and codon 44, and the mutations occured in hot spot locus of CpG dinucleotide. The Primary structure of human group IIA secreted phospholipase A2 molecule on Chinese ethnic has exactly the same structure as other ethnic groups, indicating that the molecular which plays an important role in the the host innate immune system was highly conserved among different ethnic. OBJECTIVE:To analyse the similarity between Homo sapiens phosphoilipase A2 cDNA and Leishmania infantum DNA and to understand the cause.METHODS:Searched from Gene Banks and obtained the cDNA data of human secretoryⅡA phospholipase A2 in the first step, then, by the tools of BLAST, aligned the gene fragments with every 20 bases together a group (for instance, from 1 to 20; 10 to 30;and 20 to 40···, by analogy with the others until the end of the cDNA sequence) from Homo sapiens phosphoilipase A2 cDNA to nucleotide database step by step, collecting and sorting all of the consensus sequences provide to analyze.RESULTS:There were 4 only consensus sequences between Homo sapiens phosphoilipase A2 cDNA and Leishmania infantum DNA when the length of the cDNA fragment was more or equal 17 bases, and it was differed from these that there were 9 consensus sequences between the two species when the number of the bases fragment≥16, and the sequences distributed mainly in the chromosome 29 and chromosome 30.CONCLUSIONS:There are some indenty DNA fragments between Homo sapiens phosphoilipase A2 and Leishmania infantum DNA, it may due to horizontal transfer from Leishmania to Homo sapiens. OBJECTIVE:To understand the relationship between molecular evolution and characteristics of amino acid replacement for bactericidal phospholipase A2 on primate mammals.METHODS:Amino acid sequences from Homo sapiens, Pan troglodytes and Macaca mulatta were administrated with paired comparison, and compared with group I B phospholipase A2, analyzed their location, frequency of occurrence and characteristics in their replacement of amino acid(including of their polarity, nonpolarity;acidity, neutrality, basic), respectively.Then, utilizing soft of Cn3d4.1 to build three dimensional crystal structure for their protein molecular, so as to understood their location and region of amino acid substitution on primate, made a conclusion about their change regularity, and correlated with the antibacterial activityRESULTS:There were 4,12 and 12 amino acid residues replacing among Homo sapiens, Pan troglodyte and Macaca mulatta;and the replacements of amino acid mainly ocuring in charged amino acides, especially in the positive charged ones (basic amino acid);on the other hand, amino acid replacement focused on four limited domains which play an importmant role in the enzyme function.CONCLUSIONS:Molecular evolution of group IIA phospholipase A2 is fast on primate mammals, consequently, there are more basic amino acids in IIA phospholipase A2, and it may be contributed to their powerful bactericidal activity. OBJECTIVE:To observe the bactericidal effect of the polypeptide which derived from C-terminal 26 amino acid residues of Human Group IIA phospholipase A2 (Group IIA PLA2) in vitro, and discuss its mechanism of the bactericidal effect.METHODS:According to C-terminal 26 amino acid residues sequence of Human Group IIA PLA2, and synthesized it as a piece of polypeptide P26. Incubated 6 bacterial strains (including of Staphylococcus aureus, Bacillus subtilis, Bacillus anthrax, Escherichia coli, Bacillus proteus and Bacillus pyocyaneus) solution with different concentration of polypeptide at 37℃for 2 hours in a water bath respectively. Then diluted the reaction solution and poured agar plates. After incubated for 18-24 hours in the thermostated container at 37℃, Counted the colony formed unit and calculated the bactericidal rates.RESULTS:The polypeptide P26 possessed potent bactericidal activity to the gram-positive (G+) bacteria, such as Staphylococcus aureus, Bacillus subtilis, Bacillus anthrax; weak bactericidal activity to the gram-negative (G-) bacteria, such as Escherichia coli, Bacillus proteus, Bacillus pyocyaneus.CONCLUSIONS:The polypeptide which derived from C-terminal 26 amino acid residues sequence of Human Group IIA PLA2possesses bactericidal activity. It is speculated that the polypeptide might have similar bactericidal mechanism as Human Group IIA PLA2 and the other antibacterial peptides. OBJECTIVE:To study the bactericidal activity of the polypeptide P26 and its variant due to modified and reconstructed on it, which derived from C-terminal 26 amino acid residues of Human Group IIA phospholipase A2 (Group IIA PLA2) in vitro, and analysis the cause leading to the difference of the bactericidal activity among them.METHODS:Synthesized linear polypeptide P26 corresponding to the C-terminal 26 amino acid residues sequence of Human Group IIA phospholipase A2, and then, modified it to be a cyclic polypeptide by cyclization on it, reconstructed it to be another new polypeptide by changing four basic amino acid residues of the linear polypeptide P26 into nonpolarity hydrophobic amino acid and polarity neutral amino acid.Incubated 6 bacterial species solution(including of gram-positive<G+> bacteria, such as Staphylococcus aureus, Bacillus subtilis, Bacillus anthrax;gram-negative <G> bacteria, such as Escherichia coli, Bacillus proteus, Bacillus pyocyaneus) with different concentration of polypeptide at 37℃for 2 hours in a water bath respectively. Then diluted the reaction solution and poured agar plates. After incubated for 18-24 hours in the thermostated container at 37℃, Counted the colony formed unit and calculated the bactericidal rates. RESULTS:The linear and cyclic polypeptide P26 possessed potent bactericidal activity on the gram-positive (G+) bacteria, such as Staphylococcus aureus, Bacillus subtilis, Bacillus anthrax; but weak bactericidal activity on the gram-negative (G-) bacteria, such as Escherichia coli, Bacillus proteus, Bacillus pyocyaneus. And the cyclic polypeptide P26 possess potent action on the gram-positive (G+) bacteria, but it was not for the gram-negative (G-) bacteria. Different from these, the reconstructed polypeptide P26 had the most weak bactericidal activity on the gram-positive(G+) bacteria, and none of bactericidal activity on the gram-negative (G-) bacteria.CONCLUSIONS:The linear and cyclic polypeptide P26 which derived from C-terminal 26 amino acid residues of Human Group IIA phospholipase A2possesses potent bactericidal activity against G+ bacteria. It is speculated that the more potent action of the cyclic peptide on gram-positive(G+) bacteria related to its cylic structure;when changed four basic amino acid residues of the linear polypeptide P26 into non-basic amino acid residues, bactericidal activity of the reconstructed polypeptide P26 against G+ bacteria would be decreased obviously.更多还原