B型肉毒毒素重链抗原表位的预测及表位合成肽抗原性的检测

目的:预测B型肉毒毒素蛋白抗原表位,研究表位合成肽的生物学活性。方法:利用生物信息学,结合Anthwine分析软件及网络平台,预测B型肉毒毒素蛋白重链的B细胞抗原表位。人工合成4条(P1,P2,P3,P4)表位多肽,与抗B型肉毒毒素类毒素的马血清反应;采用腹腔注射的方式用合成肽免疫BALB/c小鼠,检测免疫血清与合成肽的结合;对免疫小鼠腹腔注射B型肉毒毒素。结果:合成的多肽能与抗B型肉毒毒素的类毒素的马血清结合;多肽免疫小鼠后能产生针对多肽的抗体,其中P1、P3、P4产生的抗体对受毒素攻击的小鼠具有保护作用。结论:成功预测了抗原表位,合成的表位多肽具有较好的抗原性。     

HIV-1辅助蛋白Vpr多肽抗体的制备与鉴定

预测Vpr蛋白的B细胞抗原表位,并利用合成的B细胞表位肽制备Vpr特异性抗体。应用生物信息学技术获得Vpr蛋白共享氨基酸序列并预测其潜在B细胞抗原表位,与载体蛋白血蓝蛋白(KLH)偶联合成多肽并免疫家兔,鉴定及纯化获得的多肽特异性抗体。软件预测显示,Vpr蛋白N端的第3～19位(N)和C端的第82～95位(C)氨基酸序列为潜在B细胞抗原表位;ELISA检测抗血清中多肽特异性抗体的效价都达到1:105以上;Western-Blotting结果显示,无论对HIV-1B亚型还是CRF07_BC重组型的Vpr蛋白,其多肽N抗体和C抗体均能特异性识别;免疫沉淀结果显示,Vpr多肽N和C抗体也能特异性结合未变性的野生型Vpr或GFP-Vpr融合蛋白。利用生物信息学技术能成功预测Vpr蛋白B细胞抗原表位,免疫所获得的抗体具有较好的特异性和应用性。     

MERS-CoV棘突蛋白表位多肽疫苗设计及在小鼠体内免疫效果分析

筛选有效的免疫原,研制安全有效的疫苗,是预防中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)感染的有利武器。本研究应用生物信息学方法,预测了MERS-CoV棘突蛋白(Spike,S)上的多肽抗原表位。基于生物信息学分析结果人工合成9条多肽并偶联钥孔戚血蓝素后,分别免疫BALB/C小鼠,检测了多肽诱导的体液免疫和细胞免疫应答。结果表明,YVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGI的多肽,三次免疫小鼠后,可在小鼠体内诱导较强的IgG抗体(滴度达1∶10 000)和较高水平的抗原特异性细胞免疫应答。本研究结果为基于生物信息学预测的多肽疫苗的设计及MERS-CoV疫苗的研制提供了科学参考。     

狂犬病病毒CVS-11核蛋白B细胞线性抗原表位研究

为阐明狂犬病病毒CVS-11核蛋白B细胞线性抗原表位,本研究通过合成肽模拟B细胞线性抗原表位,采用免疫学方法对生物信息学分析获得的狂犬病病毒CVS-11核蛋白潜在B细胞线性抗原表位进行验证。结果显示,狂犬病病毒CVS-11核蛋白355~369、385~400位氨基酸序列合成肽免疫小鼠血清经间接酶联免疫吸附试验(Enzyme-linked immunosorbent assay,ELISA)检测抗多肽抗体效价达到1∶12 800以上;抗多肽抗体在免疫印迹试验(Western blot,WB)中识别变性狂犬病病毒CVS-11核蛋白抗原,在间接荧光抗体试验(Indirect fluorescent antibody,IFA)中识别感染BHK-21细胞的狂犬病病毒CVS-11核蛋白抗原。因此,狂犬病病毒CVS-11核蛋白355~369、385~400位氨基酸序列经证实为B细胞线性抗原表位。     

不同佐剂条件下Aβ多肽B细胞表位疫苗诱导产生抗体的免疫反应特性分析

目的:研究阿尔茨海默病β淀粉样肽(Aβ)B细胞表位疫苗2Aβ1-15-PADRE(Aβ-T)诱导产生抗体的免疫反应特性,并探讨不同佐剂对该疫苗免疫反应效果的影响。方法:合成了含2个Aβ42的 B细胞表位—Aβ1-15及1个辅助T细胞表位—PADRE的多肽2Aβ1-15-PADRE。采用Al(OH)₃佐剂,弗氏佐剂,Abisco佐剂,MF59佐剂分别与多肽疫苗联合免疫小鼠,并另设3个对照组:无佐剂多肽免疫组(Mock),PBS免疫组(PBS),未免疫组(Native)。结果:5组多肽免疫组小鼠均产生了针对Aβ的特异性抗体,无佐剂多肽免疫组的IgG抗体滴度最低,Al(OH)₃佐剂组,MF59佐剂组,Abisco佐剂组小鼠IgG抗体滴度较高,弗氏佐剂组IgG抗体滴度最高。斑点杂交实验结果显示5组小鼠免疫后血清与Aβ42单体反应较弱,与寡聚体反应最明显,与纤维状Aβ42几乎不反应。结论:4种佐剂均能提高多肽疫苗的免疫反应,产生高水平抗Aβ的特异性抗体。5组免疫小鼠产生的抗体均与Aβ寡聚体反应较强,与纤维状Aβ42反应较弱,表明该多肽疫苗具有良好的应用前景。     

人乳头瘤病毒-6 L1蛋白B-细胞优势表位作为多肽疫苗的研究

本研究分析了人乳头瘤病毒-6型L1外壳蛋白之B-细胞优势表位,并拟以此为基础制作表位多肽疫苗。研究中采用Goldkey和PC／Gene软件系统综合分析HPV6之L1蛋白B-细胞优势表位后,Fmoc固相合成表位多肽,通过HPLC纯化和毛细管电泳分析其纯度。与佐剂完全乳化后,免疫小鼠,进行动物水平的免疫效果评价。取免疫小鼠血清,与HPV-6 DNA阳性的尖锐湿疣患者疣体组织上清液结合,以鉴定免疫小鼠所产生抗体的特异性。发现L1蛋白第425-439位和第486-500位具有较高的免疫原性,可明显诱导小鼠血清抗体滴度升高,且该抗体与人尖锐湿疣疣体组织上清液呈阳性反应。说明所选这两个肽段为HPV6之L1蛋白的B-细胞优势表位,但诱导产生的抗体是否具有功能特异性,正在做进一步研究。     

人乳头瘤病毒-6 L1蛋白B-细胞优势表位作为多肽疫苗的研究

本研究分析了人乳头瘤病毒-6型L1外壳蛋白之B-细胞优势表位,并拟以此为基础制作表位多肽疫苗.研究中采用Goldkey和PC/Gene软件系统综合分析HPV6之L1蛋白B-细胞优势表位后,Fmoc固相合成表位多肽,通过HPLC纯化和毛细管电泳分析其纯度.与佐剂完全乳化后,免疫小鼠,进行动物水平的免疫效果评价.取免疫小鼠血清,与HPV-6 DNA阳性的尖锐湿疣患者疣体组织上清液结合,以鉴定免疫小鼠所产生抗体的特异性.发现L1蛋白第425-439位和第486-500位具有较高的免疫原性,可明显诱导小鼠血清抗体滴度升高,且该抗体与人尖锐湿疣疣体组织上清液呈阳性反应.说明所选这两个肽段为HPV6之L1蛋白的B-细胞优势表位,但诱导产生的抗体是否具有功能特异性,正在做进一步研究.     

基于纳米抗体CDR3区抗原表位展示制备生存素抗体及其鉴定

目的:通过纳米抗体CDR3区展示生存素N端表位的方式,探索纳米抗体在抗原表位展示中的作用。方法:通过基因合成方法将生存素N端起始表位(氨基酸序列1~15)插入纳米抗体CDR3区,再构建到原核表达载体pET24a中,IPTG诱导表达,用带His标签的填料纯化,获得高纯度的目的蛋白,免疫雌性BALB/c小鼠,间接ELISA检测5次免疫后的效价,用抗原偶联纯化介质纯化免疫多抗,Western印迹检测多抗特异性。结果:IPTG诱导后,目的蛋白主要以包涵体形式存在,亲和层析获得纯度大于96%的目的蛋白,包涵体经复性后免疫小鼠,效价可达1∶512000,West?ern印迹特异性检测显示免疫多抗能够特异性结合生存素。结论:纳米抗体CDR3区生存素抗原N端表位展示的方法可用于抗生存素抗体的制备,并为今后纳米抗体表位展示相关研究奠定基础。     

生长相关蛋白GAP-43多肽抗体的制备

目的对生长相关蛋白-43（growth associated proteins-43,GAP-43）进行抗原表位分析并制备兔多克隆抗体。方法通过对GAP-43 cDNA序列及氨基酸序列的结构进行生物信息分析,依据蛋白质的二级结构、亲水性、疏水性、抗原性及理化特性,经过同源性检索后,综合考虑抗体设计的其他因素,选出具有免疫活性的抗原决定簇多肽片段,采用有机固相多肽合成法合成了GAP-43的多肽片段,并与载体蛋白血蓝蛋白（KLH）偶联制备成抗原,免疫新西兰家兔。结果 ELISA测定GAP-43抗体效价为1∶32 000;Western blot检测结果显示：在分子量25kDa出现单一条带;免疫组织化学法检测显示：GAP-43蛋白在小鼠海马神经元中有表达;免疫细胞化学法检测显示：GAP-43蛋白在人神经母细胞瘤株SH-SY5Y中存在表达。结论利用生物信息学软件比较准确地预测GAP-43的抗原决定簇,并成功制备了高效价、高特异性的多肽抗体。     

小鼠子宫珠蛋白结合蛋白的生物信息学分析及其多克隆抗体制备

本文旨在制备小鼠子宫珠蛋白结合蛋白(mouse uteroglo binbinding protein,mUGBP)多克隆抗体,为后续研究工作奠定基础。通过生物信息学分析方法预测mUGBP蛋白的跨膜结构、理化特性、疏水性等因素,设计出两段多肽,分别与匙孔戚血蓝蛋白(keyhole limpet hemocyanin,KLH)交联后免疫新西兰兔,结果显示其中一个含13个氨基酸残基的多肽序列(221st～233rd)可用作抗原免疫动物,并成功获得高效价的抗mUGBP多克隆抗体。通过ELISA法检测其效价为1:108,亲和层析纯化后Westernblot检测抗体特异性,并将制备的抗体应用于免疫印迹及人和小鼠肺组织免疫组织化学和免疫荧光检测,结果显示本研究制备的抗体具有特异性,且用该抗体成功在人和小鼠气道上皮细胞和肺血管内皮细胞检测到UGBP蛋白表达。结果表明,本研究通过生物信息学方法成功预测了抗原表位,并据此预测成功制备了高效价、高特异性的抗mUGBP多克隆抗体。     

ZNF185‐derived peptide induces fertility suppression in mice

Zinc finger protein 185 (ZNF185) belongs to the ZNF family and is involved in male reproduction. However, it is unclear whether ZNF185 may be a target candidate for contraceptive vaccines. In this study, antigenic peptides derived from ZNF185 were prepared, and their immune contraceptive effects were investigated using mice. Results from enzyme‐linked immunosorbent assay (ELISAs) showed that peptide immunization induced an antibody titre increase that reached a peak in week 12. Peptide‐3 and peptide‐4 were then chosen for subsequent experiments. The results of the fertility assays showed that peptide immunization inhibited the mating and fertility rates of the mice, whereas there were no obvious changes in the number of pups per litter. Subsequently, epididymal sperm was analysed. The results demonstrated that the sperm count and sperm motility were significantly decreased in the peptide group, while the amount of abnormal sperm was significantly increased in the peptide‐3 group. The male reproductive organs were also evaluated. There were no obvious differences in testis or epididymal weights, in the diameters of the seminiferous tubules, or in the thicknesses of the seminiferous epithelium between the peptide group and the phosphate buffer saline (PBS) group. In addition, histological analysis indicated that there were no obvious pathologic changes in testis and epididymal histology in the peptide group; however, the number of spermatozoa present in the epididymal lumen of the peptide group was significantly decreased when compared with the PBS group. Our study demonstrates for the first time that peptides derived from ZNF185 may induce fertility suppression in mice without damaging reproductive organs. These peptides have the potential to be used as a male contraceptive vaccine.     

Don't pull the plug! the Drosophila mating plug preserves fertility

ABSTRACT

Mating plugs are hardened structures—typically a coagulation of seminal fluid components—that are transferred to, or formed within, the female reproductive tract of numerous animal species (both mammals and insects). Analysis of the role(s) of the mating plug in reproduction has been conducted in a wide array of diverse species. These structures have been proposed to have a multitude of functions, which include altering female re-mating rate, acting as a barrier to re-mating and being required for sperm storage or sperm movement to occur in mated females. A recent analysis of the Drosophila melanogaster mating plug has shown that proper formation of the structure is required for optimal fertility in flies: the Drosophila mating plug is required to retain the ejaculate within the female reproductive tract once mating has terminated. Here, we discuss the possible implications of the Drosophila mating plug in the fertility of this species in light of these new results.     

Defects in reproductive functions in PACAP-deficient female mice

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved neuropeptide and widely expressed in both brain and peripheral tissues, including several reproductive organs (e.g., testis and ovary). PACAP stimulates syntheses of several sexual hormones and steroids, suggesting it has possible roles in reproductive function. In this study, the role of PACAP in female reproductive functions such as fertility, mating behavior and maternal behaviors were investigated by using mice lacking PACAP (PACAP(-/-)). PACAP(-/-) females showed reduced fertility (the number of parturitions relative to the number of pairings). Mating experiments using vasectomized males revealed that mating frequency and its intervals in some PACAP(-/-) females were quite different (zero to eight times/4 weeks), whereas the frequency was relatively constant (two to three times/4 weeks) in wild-type females. In PACAP(-/-) females, maternal crouching behavior tended to decrease compared to wild-type females, although the influence of litter size on maternal behavior needs to be considered. These data suggest a role for endogenous PACAP in female reproductive processes.     

Male sexual ornament size is positively associated with reproductive morphology and enhanced fertility in the stalk-eyed fly <Emphasis Type="Italic">Teleopsis dalmanni</Emphasis>

Background  

Exaggerated male ornaments and displays often evolve in species where males only provide females with ejaculates during reproduction. Although "good genes" arguments are typically invoked to explain this phenomenon, a simpler alternative is possible if variation in male reproductive quality (e.g. sperm number, ejaculate content, mating rate) is an important determinant of female reproductive success. The "phenotype-linked fertility hypothesis" states that female preference for male ornaments or displays has been selected to ensure higher levels of fertility and has driven the evolution of exaggerated male traits. Females of the stalk-eyed fly Teleopsis dalmanni must mate frequently to maintain high levels of fertility and prefer to mate with males exhibiting large eyespan, a condition-dependent sexual ornament. If eyespan indicates male reproductive quality, females could directly increase their reproductive success by mating with males with large eyespan. Here we investigate whether male eyespan indicates accessory gland and testis length, and then ask whether mating with large eyespan males affects female fertility.     

DNA甲基化通过锌指蛋白185调控慢性粒细胞白血病细胞的增殖

锌指蛋白185(ZNF185)属于LIM结构域蛋白,参与细胞的增殖和分化,在多种肿瘤细胞中具有抑癌基因的功能.ZNF185在正常人血液系统细胞中高表达,但目前对白血病细胞的作用未见研究.采用Western blot检测人外周血中性粒细胞、急性粒细胞白血病细胞系HL-60和慢性粒细胞白血病细胞系K562细胞中ZNF185的表达,发现ZNF185在HL-60和K562细胞中的表达水平显著低于外周血中性粒细胞.为了阐明ZNF185对慢性粒细胞白血病细胞增殖的影响,从人外周血中性粒细胞克隆ZNF185编码序列,转染K562细胞,MTT检测细胞增殖,发现过表达ZNF185显著抑制K562细胞的增殖.甲基化特异PCR分析表明:ZNF185启动子在HL-60和K562细胞中高甲基化,用5-氮杂-2′-脱氧胞苷处理K562细胞,促进ZNF185的表达,显著抑制细胞增殖.研究结果表明,ZNF185启动子高甲基化导致其在K562细胞中的表达降低和细胞增殖抑制作用减弱.可能是慢性粒细胞白血病发生或发展的原因之一.     

Positive effects of testosterone and immunochallenge on energy allocation to reproductive organs

A number of studies have suggested the incompatibility of simultaneous increases in immune and reproductive functions. Other research has indicated that immune responses may be modulated depending on the relative benefits of increased survival and prospects for current and future reproduction. We tested the hypothesis that energy allocation to reproductive and other organ systems is not affected by testosterone level and energy expenditure on immune functions. Adult male white-footed mice (Peromyscus leucopus) with or without elevated testosterone levels and with or without immunochallenges were tested. Testosterone treatment was associated with reduced humoral immune response indicating immunosuppressive effects, reduced masses of gastrointestinal organs, reduced corticosterone level, increased kidney and seminal vesicle masses, and increased hematocrit. Immunochallenge was associated with increased resting metabolic rate and testes and seminal vesicle masses. Reproductive organ masses were greatest in immunochallenged mice with exogenous testosterone. Simultaneous increases in energy allocation to immune and reproductive structures may be an adaptive response that would enhance survival and current prospects for reproduction.     

锌指蛋白185 在小鼠睾丸支持细胞中的表达与定位

目的:检测锌指蛋白185在小鼠睾丸支持细胞中的表达,探讨其与精子发生的关系。方法:提取睾丸和支持细胞总RNA,经半定量RT-PCR法检测ZNF185的转录水平;提取睾丸和支持细胞的蛋白质,利用Western blot分析ZNF185的表达量;制备支持细胞爬片,采用免疫荧光技术检测ZNF185的定位。结果:(1)半定量RT-PCR结果显示:在睾丸支持细胞中扩增出ZNF185基因条带。(2)Western blot结果显示:ZNF185在支持细胞中的表达量显著低于在睾丸组织中的表达量。(3)免疫荧光结果显示:ZNF185主要定位于支持细胞胞质中。结论:ZNF185可能参与支持细胞结构与功能的调控,进而影响精子发生过程。     

A delay in age at first mating results in the loss of future reproductive potential via apoptosis

Summary Women who delay childbearing risk subfertility. However, this loss of fertility is not a simple function of aging. Women who have had children early in life tend to maintain fertility longer, measured as a later age at menopause. But why should otherwise healthy women lose reproductive capacity? Loss of fertility independent of senescence, menopause, has been approached from two perspectives: evolution and development. Evolutionary biologists focus on how natural selection favors survival after reproductive ability has ceased, whereas reproductive biologists examine mechanisms by which women lose fertility with age and factors that influence the rate of reproductive aging. Combining mechanistic studies with evolutionary theory should allow us to define principles of the evolution of postembryonic development of ovaries, including the role of reproductive timing relative to sexual maturation. Achieving this will require identifying appropriate, and more experimentally tractable, taxa in which to study how early reproductive events influence lifetime fertility. We work with an invertebrate species, the cockroach Nauphoeta cinerea, with a complex reproductive biology in which females experience reproductive cycles, give live birth, and show age‐related decline in fertility. Thus, N. cinerea provides an opportunity to use an experimental approach to examine mechanisms by which females lose reproductive potential as they delay reproduction. Our results demonstrate that the loss of both oocytes ready for fertilization and future oocytes in females that delay mating is because of apoptosis. We suggest that loss of fertility because of delayed mating may originate in a nonadaptive response in control of apoptosis through mistiming of reproduction.     

Long intergenic non-protein coding RNA 00858 participates in the occurrence and development of esophageal squamous cell carcinoma through the activation of the FTO-m6A-MYC axis by recruiting ZNF184

ObjectivesWe aimed at probing impact of LINC00858 on esophageal squamous cell carcinoma (ESCC) progression via ZNF184-FTO-m⁶A-MYC axis.MethodsExpression of related genes (LINC00858, ZNF184, FTO, and MYC) was detected in ESCC tissues or cells and their relationships were assessed. After expression alterations in ESCC cells, cell proliferation, invasion, migration, and apoptosis were detected. Tumor formation in nude mice was conducted.ResultsLINC00858, ZNF184, FTO, and MYC were overexpressed in ESCC tissues and cells. LINC00858 enhanced ZNF184 expression to upregulate FTO, which augmented MYC expression. LINC00858 knockdown diminished ESCC cell proliferative, migratory, and invasive properties while elevating apoptosis, which was negated by FTO overexpression. FTO knockdown exerted similar functions of LINC00858 knockdown on ESCC cell movements, which was annulled by MYC upregulation. Silencing LINC00858 repressed tumor growth and related gene expression in nude mice.ConclusionsLINC00858 modulated MYC m⁶A modification via FTO by recruiting ZNF184, thus facilitating ESCC progression.     

Effects of p-nonylphenol and resveratrol on body and organ weight and in vivo fertility of outbred CD-1 mice

The aim of this study was to analyse the multigenerational effects of para-nonylphenol (NP) and resveratrol (RES) on the body weight, organ weight and reproductive fitness of outbred CD-1 mice. The data indicate that in male mice, NP had an effect on the weight of selected reproductive organs and the kidneys in the parental (P) generation males. Effects on selected reproductive organs, the liver and kidneys in the F1-generation males were also seen. In females, effects of NP on body weight and kidney weight were seen in the P generation, but no effects on any measured parameter were seen in the F1 generation. RES had no effect on body weight but did have some effect on selected male and female reproductive organs in the P generation. RES altered the spleen and liver weights of P-generation males and the kidney weight of F1-generation males. Acrosomal integrity (using a monoclonal antibody against intra-acrosomal sperm proteins) was assessed for both generations of NP- and RES-treated mice. A significant reduction in acrosomal integrity was seen in both generations of NP-treated, but not in RES-treated, mice. Fewer offspring were observed in the second litter of the F2 generation of mice treated with NP; no similar effect was seen in RES-treated mice. The litter sex ratio was not different from controls. Unlike RES, NP had a negative effect on spermatogenesis and sperm quality with a resultant impact on in vivo fertility.