【作者】 孙树民；

【导师】 刘明远；

【作者基本信息】 吉林大学 ， 预防兽医学， 2006， 硕士

【摘要】 旋毛虫病是由旋毛虫(Trichinella spiralis)引起的一种呈全球性分布的人兽共患寄生虫病,严重威胁着人类的身体健康。本研究利用分子生物学及其技术,分离肌幼虫抗原基因,目的是为旋毛虫病诊断抗原的寻找奠定基础。本研究成功的克隆鉴定出旋毛虫肌幼虫的抗原基因p53和p45 ,并以基因工程菌表达,为进一步将旋毛虫不同时期的抗原基因进行重组,表达复合基因重组抗原,建立敏感、特异的旋毛虫病免疫学检测方法奠定基础。p53 kDa抗原基因和p45 kDa抗原,全长分别为1176 bp和810bp,有完整的开放阅读框。GenBank数据库BlastN同源性分析表明,与p53和Tryp-SPc基因序列(TSU25127)和(AAA20539.1)高度同源,同源性分别为(98%)和(99%)。利用PCR技术,从旋毛虫肌幼虫cDNA文库中调取p53和p45抗原基因,得到重组质粒并扩增到不含信号肽序列目的基因片段,重组到原核表达载体pET-28a中。用IPTG诱导培养重组表达菌,对表达产物进行SDS-PAGE分析,结果显示,经IPTG诱导后重组菌体裂解产物与对照菌相比分别出现1条相对分子量约为48 kDa和30 kDa蛋白条带。研究表明二基因在旋毛虫肌幼虫和成虫期均有表达。Western-blot检测表明,两基因重组蛋白可以被旋毛虫感染猪血清识别,具有很好的反应原性。本实验成功的从旋毛虫肌幼虫cDNA文库中调取p53和p45抗原基因,获得旋毛虫肌幼虫抗原基因;将两个基因克隆到原核表达载体上,并在大肠杆菌中进行高效表达;并将其表达的融合蛋白进行Western-blot检测,并对其序列进行分析和蛋白结构的预测,达到预期结果。更多还原

【Abstract】 Trichinellosis was a global zoonotic parasitic disease caused bynematode. It was still prevalence in China. More than 150 mammals couldbe infected besides human . Trichinellosis could not only cost great expensesin animal industry but also endanger to human health .The multiplicityTrichinella spiralis antigens made a difficulty for a large-scale reproducingin vitro,immunodiagnosis and prevention .Trichinella spiralis muscle larvaeis infected with host, experience four exuviations to develop into imago in30h.Consequencely,it gave birth to newborn larvae in 96h and newborn larvaeinvaded into all skeletal muscle by lymph and blood. Finally, newborn larvaegrew up infective muscle larvae, which could survival in animal and humanbody for long term. Muscle larvae take in host nutrition and endangered tohost health. Recently, Trichinella spiralis study only focus on antigen, butits antigen is multiplicity. Though ES was used of immunodetection in trichiniasisideally, however, epitope of ES had nonspecific reaction with factor of human serum.Meanwhile, it could lead to a false positive diagnosis and there were difficulty forlarge-scale preparation. Molecular biology and bioinformation software was appliedfor p53 and p45 from cDNA of Trichinella spiralis muscle larvae to clone,expression and identification in the study .It found in trichinelliasis diagnosisantigen of large scale preparationp53 and p45 was gotten from cDNA library of Trichinella spiralis and itspositive clone was sequenced. The sequence was analysised by molecularbiology software.The result show that 1176bp of p53 coda was successfulcloned,encoded 391 amino acids which was 98% isogeny with TSU25127.Coproteinhomology analysis indicated p53 cloned sequence was 98% homology with 53kDaantigen of Trichinella spiralis. p53 contained three N-glycosylation site. P45 cDNAand encoded sequence analysis showed that cloned p45 cDNA was 810bp and encoded270 amino acids. The sequence isogeny was 99% with U01847of GenBank gp45, was99% with AAA20539.1 of Trichinella spiralis 45 kDa antigen. P45 contained one N-glycosylation site. Trichinella spiralis 53kDa ES antigen could expression duringmuscle larva and adult stage and was candidate diagnosis antigen. Analysis of itssecond order construction and sequence would bring hope for cell predominance shortpeptide study and provide theory support for high performance recombination proteinwith strong antigenicity immunogenicity. 45kDa ES antigen of Trichinella spiraliscontained conservation domain, gene polymorphism that expressed duringmuscle larva and adult stage. It could have a multigene family, its function was similarto serine protease. Though it losed enzymatic active center during evolutionaryprocess, however it still remained antigen activation. Recombinant plasmid wasobtained from cDNA library to amplication and gotten p53 and p45 gene fragmentwithout signal peptide .The fragment was combinated with pET-28a. pET-28a-p53 andpET-28a-p53 was translated in Nova blue and BL21(DE3)competent cell. Plastid wasextract and identified by enzyme cutting and sequencing. Engineer bacterium wasinduced and trained in IPTG, analysis in SDS-PAGE. The result showed that 48kDaprotein appear in cleaved product lane of induced bacterium, it match to fusion proteintheory value. Lamellar scan result indication that with inducing time prolonging,protein expression amount would also increased and reach summit in 4h.Propotion ofinterest protein and thallus total protein was 37.077%. The study result showed thatp53 and p45 gene would express during larva and adult stage.Western-blotting result showed that recombination antigen could was recognizedby pig, mouse and immunization mice serum of recombination antigen withTrichinella spiralis infection history. It illuminated that host withTrichinella spiralis infection history and immunization of recombination antigencould stimulate host immunity system and provoke antigenic specific antibodyresponse. Recombination antigen posses better antigen city thus it was base ontrichinelliasis diagnosis and large-scale preparation. The study successful gotten p53and p45 gene of Trichinella spiralis muscle larva and express it in engineerbacterium. It promoted p53 kDa and p45 kDa antigen of Trichinella spiralismuscle larva to become candidate gene, antigen gene recombination inTrichinella spiralis every growth stage, expression of polygenicrecombination antigen and make foundation for establishment of sensitiveand specific immunology detect method.更多还原