旋毛虫新生幼虫cDNA文库构建及其特异性基因的筛选与鉴定

构建旋毛虫新生幼虫cDNA文库并对旋毛虫新牛幼虫期特异件基因进行筛选与鉴定,筛选出期特异性的基因片段.用λZAPⅡExpress载体成功地构建了旋毛虫新生幼虫的cDNA文库,并用放射性同位素a-38P标记cDNA探针对筛选出的阳性克隆进行鉴定.所构建的cDNA文库容量为1.92×106,重组效率为98.6%,所有的克隆片段都在0.5～2.0 kb之间,筛选获得7个阳性克隆,其大小在1.4 kb左右;用放射性同位素a-32P标记cDNA探针后,与旋毛虫成虫、肌幼虫、新生幼虫及成虫/新生幼虫cDNA文库质粒DNA进行Southern印迹杂交,结果与新生幼虫和成虫、新生幼虫cDNA文库质粒DNA有杂交而与成虫、肌幼虫cDNA文库质粒DNA不杂交.该基因是新生幼虫期所特有的cDNA片段.     

旋毛虫新生幼虫差减cDNA文库的构建及其步筛选

利用差减杂交（减法杂交，subtractive hybridization)技术，以旋毛虫新生幼虫（newborn larvae,NBL)的cDNA作为试验方（tester)，以肌幼虫＋成虫的cDNA作为驱动方（driver)，制备新生幼虫差减cDNA,利用T载体构建NBL差减cDNA文库，获克隆株90个，以试验方的差减cDNA第2次PCR产物＋未差减cDNA为试验方探针，以驱动方的差减cDNA第2次PCR产物＋未差减cDNA为驱动方探针，在NBL差减cDNA文库中初筛NBL的期特异性克隆，获初筛克隆24个。这些初筛斯异性克隆进一步用Southern blot确认鉴定，获直阳性期特异性克隆2个（NBL SSC1，NBL SSC2）.DNASIS以及Blaster的分析结果显示，这2个克隆为旋毛虫的2个新基因，其中NBL SSC1编码糖蛋白，NBL SSC2编码丝氨本蛋白酶，本试验为旋毛虫期特异性基困全长序列的调取，分析，鉴定以及强保护性抗原基因的筛选奠定了基础。     

旋毛虫5日龄成虫期特异性基因片段的克隆及鉴定

利用差减抑制杂交（SSH）技术，以旋毛虫5日龄成虫的cDNA为试验方（tester），以肌幼虫＋3日龄成虫的cDNA为驱动方（driver），制备5日龄成虫差减cDNA，并与pT-Adv载体相连接，转入大肠埃希氏菌TOP 10F。以试验方的差减cDNA PCR产物＋未差减cDNA为试验方探针，以驱动方的差减cDNA PCR产物＋未差减cDNA为驱动方探针，对5日龄成虫差减cDNA克隆进行初步筛选，对筛选到的阳性克隆进一步用southern bolt进行鉴定，并对其测序，进行序列分析。结果表明，获得1个大小为464bp的旋毛虫5日龄成虫期特异性cDNA片段，经BLAST软件分析表明，该序列是1个未见报道的旋毛虫基因序列片段，可能编码1种表皮胶原蛋白。这为旋毛虫5日龄成虫期特异性基因全长序列的钓取、分析及鉴定奠定了基础。     

旋毛虫FYVE锌指结构蛋白基因的克隆及表达

用旋毛虫新生幼虫差减cDNA文库中的T54克隆作为核酸探针，对旋毛虫成虫和新生幼虫混合cDNA文库进行筛选，获得1个全长1464bp的cDNA分子，该cDNA含有1个1290bp的开放阅读框架（ORF），Blastn同源性分析表明，与其它已知生物基因序列无明显的同源性，为一新的cDNA。该ORF编码1个429个氨基酸残基组成的多肽，其分子质量理论推导值为49．9ku，等电点为5．6，在12－14及103－105氨基酸残基处分别有2个糖基化位点NLS及NVS，在C末端344－409氨基酸残基处有1个FYVE锌指结构域（Profile PS50178)。Blastp同源性分析表明，与广东管圆线虫（Angiostrongylus cantonensis)66.0ku蛋白（gi/1743430)同源性最高，为39．0％。在此基础上对旋毛虫FYVE锌指结构重组蛋白进行了表达，表达蛋白占菌体蛋白的24％。     

旋毛虫新生幼虫期特异性T314全长cDNA的克隆及表达

利用PCR将旋毛虫新生幼虫期特异性全长T314cDNA的信号肽祛作后重组到原核表达载体pET-28a。将重组质粒pET28-T314转入克隆菌DH5a和表达菌DE3，分别提取质粒进行酶切，测序鉴定。用KIPTG诱导培养重组表达菌DE3，做菌体裂解物外源表达产物的SDS－PAGE分析。将重组质粒表达的融合蛋白免疫家兔制备抗血清，采用Western blot检测T314cDNA在旋毛虫不同发育时期的表达情况及其天然表达产物的相对分子质量。测序结果显示：外源T314cDNA的PCR产物在重组质粒pET28-T314中阅读框架正确；SDS－PAGE分析结果显示：经IPTG诱导后转化菌DE3的裂解产物与对照菌相比出现了1条相对分子质量约为39000的新条带，大小与外源cDNA融合蛋白的理论推导值38800相符；Western blot检测结果显示：T314cDNA的天然表达产物仅存在旋毛虫新生幼虫，不存在于成虫与肌幼虫，且相对分子质量大小与其在原核重组质粒中表达产物相同。     

旋毛虫肠道1期幼虫抗原性基因的筛选与分析

筛选旋毛虫肠道1期幼虫抗原性基因。利用λZAP载体构建旋毛虫肠道1期幼虫cDNA文库。用感染旋毛虫26 d猪血清对其进行免疫学筛选,对筛选出的阳性克隆进行测序与分析;应用RT-PCR方法对编码p46 000蛋白强阳性克隆基因在不同发育时期虫体转录情况进行检测。成功构建了旋毛虫肠道1期幼虫cDNA文库,其库容量为1.5×106pfu,重组率为95%,插入片段在400 bp~2 000 bp之间,扩增后文库滴度为1.5×1012pfu/mL。筛选出阳性克隆33个,序列分析表明,有20个克隆为同一已知基因,称为旋毛虫p46 000蛋白,编码旋毛虫半胱氨酸蛋白酶抑制剂,且在筛选过程中反应信号均较强;还发现了一些旋毛虫新基因,如编码Ubc蛋白,GM13181蛋白,Rieske硫铁蛋白1等。本研究获得了一个高丰度、反应原性较强抗原基因,其编码蛋白为半胱氨酸蛋白酶抑制剂。     

旋毛虫丝氨酸蛋白酶基因的克隆与分析

以旋毛虫感染猪血清为抗体探针,对旋毛虫3日龄成虫cDNA文库进行免疫筛选,将获得的阳性克隆进行测序,用分子生物学软件对所得cDNA序列进行了分析。序列分析结果显示,阳性克隆Zh68cDNA全长为1372bp,含有1个1287bp的完整的开放阅读框架,编码由429个氨基酸组成的多肽,理论分子质量为47．5ku,等电点为8．45。SMART分析表明,1～18位氨基酸为信号肽序列,37～277位氨基酸为典型的丝氨酸蛋白酶胰蛋白酶类结构域,其中His88、Asp142、Ser233为催化中心的3个主要氨基酸残基,78～80位氨基酸为N-糖基化位点（NCS）,另外还有6个保守的Cys形成二硫键。BLASTn同源性分析表明,与其他生物丝氨酸蛋白酶的基因序列无明显的同源性,为1个新的cDNA分子。BLASTp蛋白质同源性分析表明,与丝氨酸蛋白酶的同源性为30％左右。Southern—blot杂交表明,此基因在旋毛虫基因组中属于多基因家族,具有基因多态性。cDNA的PCR结果表明,此基因在旋毛虫肌幼虫、新生幼虫及成虫期均有表达。     

家猪Fosmid基因组文库的构建

本研究采用蛋白酶K和苯酚抽提法提取基因组DNA，通过物理方法随机剪切DNA，经T₄DNA聚合酶和T₄多聚核苷酸激酶末端修饰，琼脂糖凝胶电泳回收大小为25~40 kb的片段，与pCC1FOS载体连接，噬菌体包装蛋白体外包装，转染大肠杆菌EPI300，构建了家猪Fosmid基因组文库。文库容量为8.50×10⁹CFU/ml，平均插入片段大小约25 kb，用家猪MC1卫星DNA做探针对文库进行初步筛选，获得含该卫星DNA的阳性克隆，该文库的构建为进一步筛选并研究猪卫星DNA序列奠定了基础。     

旋毛虫新生幼虫p46 000抗原基因的克隆及序列分析

应用旋毛虫感染猪血清，对旋毛虫新生幼虫cDNA文库进行了免疫筛选。对阳性克隆pBK-cMV-WN10的序列分析结果表明。cDNA全长为1352bp。含有1个1218bp的完整的开放阅读框架(ORF)，编码的多肽由406个氨基酸残基组成，其相对分子质量理论推导值为45900，等电点为5．43，N末端的信号肽及糖基化位点(NCS)表明其可能为分泌性糖蛋白，氨基酸序列19～156与158～295为重复区域，相似性为74％．C末端有1个半胱氨酸蛋白酶抑制剂结构域，但旋毛虫p46000抗原与其他线虫的半胱氨酸蛋白酶抑制蛋白结构有很大差异，可能已经失去半胱氨酸蛋白酶抑制蛋白的功能。PCR结果显示。从旋毛虫新生幼虫、肌幼虫、3日龄成虫和5日龄成虫cDNA中均扩增出此基因，表明此基因在旋毛虫各个时期均有表达。     

中国旋毛虫分离株成虫和新生幼虫基因文库的构建及筛选

利用λＺＡＰⅡ载体构建了中国分离株Ｔｒｉｃｈｉｎｅｌａｓｐｉｒａｌｉｓ及Ｔｒｉｃｈｉｎｅｌａｎａｔｉｖａ成虫和新生幼虫ｃＤ－ＮＡ基因文库,检测结果表明,所克隆的ｃＤＮＡ片段分布在０．２～２．０ｋｂ之间,表明２个ｃＤＮＡ基因文库对ｍＲＮＡ的覆盖面很大。利用ＲＡＰＤ技术,以肌幼虫基因文库作对照,采用７０个单引物及２０对复合引物对以上２个文库进行筛选扩增,结果：（１）共获得２条Ｔ．ｓｐｉｒａｌｉｓ及４条Ｔ．ｎａｔｉｖａ的成虫与新生幼虫特异性基因片段,鉴于成虫与新生幼虫是旋毛虫的２个侵袭性阶段,其所特有的特异性基因片段极有可能就是这２个时期虫体的侵袭性因子基因,即所要寻找的强保护性抗原基因,从而为后续研究奠定了基础;（２）发现１条Ｔ．ｓｐｉｒａｌｉｓ种特有的基因片段,这一特异性基因片段为虫种鉴定提供了物质条件;（３）发现旋毛虫同种不同分离株（中国分离株及法国分离株）间ｃＤＮＡ文库扩增图谱完全相同,进一步证实了旋毛虫种内基因的低流动性     

多拉菌素浇泼剂对小鼠旋毛虫的疗效研究

研究了多拉菌素浇泼剂对小鼠旋毛虫各个时期的驱杀效果。采用多拉菌素浇泼剂按5mg·kg^-1·bw^-1为小鼠背部浇泼给药一次,对小鼠旋毛虫成虫、移行期幼虫和包囊期幼虫进行驱杀。结果表明。多拉菌素浇泼剂对旋毛虫成虫的杀虫效果最好,杀虫率可达99．5％,其次是旋毛虫的移行期幼虫,杀虫效果达96．62％,而对包囊期幼虫的杀虫效果最差,只有31．98％。多拉菌素浇泼剂治疗小鼠旋毛虫的成虫和移行期蚴虫疗效显著,而对包囊期幼虫效果不明显。本研究为该药进一步的广泛应用于家畜提供理论与试验依据。     

Molecular cloning of a cDNA encoding a putative cuticle collagen of Trichinella spiralis

A 5-day-old adult stage-specific cDNA fragment from Trichinella spiralis was identified by suppression subtractive hybridization and was used as a probe to screen the cDNA library. The cDNA sequence coding for a putative T. spiralis cuticle collagen was isolated. The cDNA encoded an open reading frame of 343 amino acid residues with molecular weight of 35.1 k Da. The deduced protein contained an N-terminal signal peptide, a nematode cuticle collagen N-terminal domain and a collagen triple helix repeat domain. Searches in GenBank using BLASTP showed up to 47% identity to cuticle collagens from other nematodes. Southern blot analysis of genomic DNA indicated this gene was present as a single copy in T. spiralis genome.     

用地高辛标记的核酸探针检测猪伪狂犬病毒野毒感染的研究

利用PCR技术从带有伪狂犬病毒（PRV）gE基因的重组质粒pMD18-T-gE中扩增回收约304bp大小的片段,并制备出地高辛标记的gE基因核酸探针。特异性检测结果表明,该探针能与重组质粒DNA发生特异性杂交,而与对照的PRVBartha-k61株疫苗毒DNA、猪细小病毒（PPV）DNA、猪圆环病毒（PCV）DNA、猪繁殖与呼吸综合征病毒（PRRsV）cDNA、猪瘟病毒（CSFV）cDNA的杂交反应均为阴性;敏感性检测结果表明,该探针对PRV野毒的最低检出量为4pg。应用该探针对11份繁殖障碍病料进行了杂交检测,共检出4份阳性病料,该结果与PCR检测结果一致,表明该核酸探针可用于猪伪狂犬病野毒感染的临床诊断。     

Differential detection of Trichinella papuae, T. spiralis and T. pseudospiralis by real-time fluorescence resonance energy transfer PCR and melting curve analysis

Trichinellosis caused by nematodes of Trichinella spp. is a zoonotic foodborne disease. Three Trichinella species of the parasite including Trichinella spiralis, Trichinella papuae and Trichinella pseudospiralis, have been etiologic agents of human trichinellosis in Thailand. Definite diagnosis of this helminthiasis is based on a finding of the Trichinella larva (e) in a muscle biopsy. The parasite species or genotype can be determined using molecular methods, e.g., polymerase chain reaction (PCR). This study has utilized real-time fluorescence resonance energy transfer PCR (real-time FRET PCR) and a melting curve analysis for the differential diagnosis of trichinellosis. Three common Trichinella species in Thailand were studied using one set of primers and fluorophore-labeled hybridization probes specific for the small subunit of the mitochondrial ribosomal RNA gene. Using fewer than 35 cycles as the cut-off for positivity and using different melting temperatures (T(m)), this assay detected T. spiralis, T. papuae and T. pseudospiralis in muscle tissue and found the mean T(m) ± SD values to be 51.79 ± 0.06, 66.09 ± 0.46 and 51.46 ± 0.09, respectively. The analytical sensitivity of the technique enabled the detection of a single Trichinella larva of each species, and the detection limit for the target DNA sequence was 16 copies of positive control plasmid. A test of the technique's analytical specificity showed no fluorescence signal for a panel of 19 non-Trichinella parasites or for human and mouse genomic DNA. Due to the sensitivity and specificity of the detection of these Trichinella species, as well as the fast and high-throughput nature of these tools, this method has application potential in differentiating non-encapsulated larvae of T. papuae from T. spiralis and T. pseudospiralis in tissues of infected humans and animals.     

旋毛虫新生幼虫期特异性T668全长cDNA的克隆及序列分析

利用旋毛虫新生幼虫期特异性 c DNA片段 T6 6 8- SS2作为核酸探针 ,在新生幼虫 c DNA文库中筛选出 10个阳性克隆。序列测定及分子生物学软件分析表明 ,克隆 G10 - 6的 c DNA片段全长 16 0 0 bp,含 12 90 bp的开放阅读框架 ,编码 1个由 4 30个氨基酸残基组成的多肽 ,其相对分子质量理论推导值为 4 6 84 0 ,等电点 (PI)为 9.6 5。主导氨基酸为Ser(11.39% )和 Thr(10 .93% )。开放阅读框架中具有丝氨酸蛋白酶保守功能区及酶活性位点结构 ,其 N末端的信号肽及 2个糖基化位点表明 ,其为分泌性糖蛋白 ,预示其可能在细胞外起着重要作用。DNA同源性分析表明 ,该 c DNA为一新的 c DNA分子。     

Expression of LacZ gene in canine muscle by intramuscular inoculation of a plasmid DNA

DNA immunization induces systemic humoral and cellular immune responses to the antigen encoded by cDNA in a plasmid DNA. In the present study, a plasmid DNA encoding cDNA of beta-galactosidase (beta-gal), pCAGGS-lacZ, was inoculated intramuscularly to a healthy dog in order to evaluate location and duration of the gene expression. On day 7, the plasmid DNA was found by PCR in the muscle where the plasmid was injected. Furthermore, beta-gal expression was detected in the same muscle sample by beta-gal staining. However, the plasmid DNA was not detected in any samples collected on days 14, 21 and 28. The present results suggest that duration of the gene expression of beta-gal by the plasmid DNA is limited in the muscle in dogs and an efficacy for a gene expression should be evaluated depending on the gene inserted in the plasmid DNA for immunotherapy.     

禽流感病毒核蛋白基因在重组杆状病毒中的表达

利用ＲＴ－ＰＣＲ方法成功地扩增了我国禽流感病毒分离株Ａ／Ｘｉｎｇｊｉａｎｇ／１／９６（Ｈ１４Ｎ５）的核蛋白（ＮＰ）基因,其限制性内切酶图谱和核苷酸序列与鸭源的标准Ｈ１４Ｎ５毒株几乎完全一致,与其它毒株则有较大差异,说明该鸡源分离株与鸭源毒株有非常近的亲缘关系。将ＮＰ基因定向克隆到杆状病毒转移载体ｐＶＬ１３９３中,再与杆状病毒线性ＤＮＡ（ＢＡＣ－Ｎ－ＢｌｕｅＤＮＡ）共转染于Ｓｆ９昆虫细胞中,经过三次蚀斑筛选,获得重组病毒ｒＢ２。用其细胞表达产物裂解后作ＳＤＳ－ＰＡＧＥ蛋白电泳、Ｗｅｓｔｅｒｎ－ｂｌｏｔ和ｄｏｔ－ＥＬＩＳＡ,结果表明ＮＰ基因在杆状病毒系统中获得了表达。同时用表达产物作琼脂扩散试验,结果表明表达产物与现行标准禽流感琼扩抗原具有相同的生物学活性。     

Diagnostic complementary DNA probes for genome segments 2 and 3 of epizootic hemorrhagic disease virus serotype 1

Potential diagnostic complementary DNA (cDNA) clones of gene segments 2 and 3 from epizootic hemorrhagic disease virus serotype 1 (EHDV-1) have been produced. Individual segments of EHDV-1 were isolated, denatured with methylmercury hydroxide, and polyadenylated. The polyadenylated RNA was reverse-transcribed and self-hybridized into duplex structures, and the incomplete ends were repaired. The resulting product was then cloned into the plasmid vector pBR322, using the complementary tailing method. Two clones, 1 from segment 2 (E1-2-10) and 1 from segment 3 (E1-3-16) were isolated, colony-purified, and characterized by cDNA/RNA blot hybridization and endonuclease restriction analysis. The cDNA clones of RNA segment 3 of EHDV-1 cross hybridized with the corresponding segment of EHDV serotype 2 by results of cDNA/RNA blot hybridization, but not with RNA of bluetongue virus serotypes isolated in the United States. After cDNA/RNA dot-blot hybridization analysis of 17 EHDV field strains, the segment-2 clone was found to be serotype-specific, whereas the segment-3 clone was serogroup-specific.     

旋毛虫肌幼虫分泌性蛋白P49基因的克隆与表达

通过设计、合成引物，以旋毛虫RNA为模板，用RT—PCR法扩增出旋毛虫P49序列，并进行了序列测定。将P49基因亚克隆至表达载体pET—28b中，转化大肠杆菌BL21感受态细胞，经IPTG诱导表达，作SDS—PAGE及Western blot分析。结果表明，PCR法扩增出P49序列，其大小约为960bp，将构建的重组质粒pGEM—P49进行序列测定表明其与Genbank中的P49序列具有高度的同源性。成功构建了重组表达载体pET—P49；SDS—PAGR及Western blot分析表明，表达产物分子量约为38kDa，约占菌体总蛋白的7％左右，且能被感染旋毛虫猪阳性血清所识别。     

Detection of bovine viral diarrhea virus by spot hybridization with probes prepared from cloned cDNA sequences

A cytopathic strain of bovine viral diarrhea virus (BVDV) was purified from infected cell culture fluids by isopycnic density-gradient centrifugation. Genomic RNA was extracted and tailed with adenine residues at the 3' end with poly-A polymerase. Double-stranded complementary DNA (cDNA) was synthesized, using the poly-A-tailed RNA as a template and oligo-dT as a primer, and then cloned into the pUC9 plasmid. Virus-specific cDNA sequences, varying in length from 0.5 to 2.5 kilobases (kb), were obtained. One BVDV-specific sequence of cloned cDNA, 1.1 kb in length and with an internal Pst I restriction endonuclease cleavage site, was selected for use as a probe. The cloned cDNA insert was removed from the plasmid either with or without flanking plasmid sequences and labeled with 32P-nucleotides by nick translation for use as hybridization probes for BVDV. The performance of probes of smaller fragments of the insert was compared to that of the intact sequence in hybridization assays. In addition, 2 methods of specimen preparation were compared to establish optimum parameters for hybridization. The hybridization assay was 10-100 times more sensitive than infectivity assays for BVDV in infected cell cultures. Freezing of specimens reduced by 10-fold the sensitivity of hybridization for BVDV target sequences. The probes prepared from the cloned cDNA hybridized with all cytopathic and noncytopathic BVDV strains tested but not with uninfected cell cultures, cellular ribosomal RNA, bovine coronavirus, bluetongue virus, or bovine adenovirus 3. Probes prepared with native plasmid DNA did not hybridize with BVDV or uninfected cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)