疥螨原肌球蛋白基因的克隆、表达与蛋白定位研究
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摘要
疥螨病是一种常见的人兽共患寄生虫病,具有高度传染性,能导致人和动物剧痒及各种类型的皮炎,是一种顽固性、接触性、传染性皮肤病。长期药物治疗疥螨病存在药物残留、环境污染等问题。目前尚无可用于免疫防治的疥螨疫苗。尽管寄生虫疫苗的研制较为困难,但相信随着分子生物学的不断发展,疥螨基因文库的不断完善,疥螨基因工程疫苗将会成为防治疥螨病最有前景的方法之一
本研究参考GenBank中收录的粉尘螨(D17682)、屋尘螨(AF016278)和痒螨(AM114276)原肌球蛋白(Tropomyosin)基因序列设计引物,对提取的疥螨总RNA进行RT-PCR扩增与克隆,测序结果得到了855bp的目的基因片段。通过对该基因核苷酸序列进行分析,与已报道的粉尘螨、屋尘螨和痒螨原肌球蛋白(Tm)基因的序列同源性分别为87.60%,85.26%和85.03%。预测其表达的重组蛋白大小约为50.90kDa,理论等电点值为pI=4.43,具有很强的亲水性。推导的化学式为C[39] H2318N412O4878S10,带负电荷。该蛋白不存在信号肽切割位点,也不存在跨膜结构域,是一种膜外蛋白。
将目的片段插入pET32a(+)表达载体上,将测序正确的重组质粒转化大肠杆菌BL21(DE3),用浓度为1.0mM/L的IPTG诱导表达了疥螨原肌球蛋白,通过SDS-PAGE分析表明,在相对分子质量约50kDa处有明显的表达条带。进一步优化诱导表达条件,结果表明该重组蛋白在37℃温度下,IPTG浓度为0.4mol/L,诱导4h时基本达到最大表达量。用兔疥螨阳性血清作为一抗进行免疫印迹反应,发现重组表达蛋白可与兔疥螨阳性血清反应,产生蛋白免疫印迹带,说明表达的重组蛋白具有抗原性。
将纯化的重组蛋白与弗氏佐剂混合制备乳剂苗,用其免疫家兔,获得高免血清,并通过免疫组织化学SP技术,对疥螨原肌球蛋白进行组织定位,试验结果表明该蛋白主要在疥螨口器和四肢的肌肉,躯体表皮以及内脏器官表膜中表达。
Sarcoptic mange is a common and highly contagious human zoonotic parasitic disease, highly contagious, which can cause itching and various types of dermatitis, and is a stubborn, contagious skin disease. Over the years, medicines are still the main control of Sarcoptic mange, yet can be used for prevention and treatment of vaccine immunization. The traditional treatment of scabies is usually drug, it is not only ineffective, but also there are drug residues and environmental pollution. Although the development of parasite vaccine is difficult, especially genetic engineering vaccine, but the scabies treatment of genetic engineering vaccine will become the most promising methods with the continuous development of molecular biology, gene library of scabies mites continue to improve.
Here scabies mites from the rabbit were collected, and a pair of primers was designed referencing GenBank. The 855 bp DNA fragment was amplified by polymerase chain reactions,encoding a 285 amino acid residue polypeptide. By blasting the homologous sequence in GenBank databases, the gene which encoded a predicted protein with 87.60%,85.26% and 85.03% identity to the Dermatophagoides farinae allergen Der p10, Dermatophagoides pteronys sinus allergen Der p10 and Psoroptes ovis allergen P. ovis 10 respectively. The molecular mass of polypeptide was 50.90 kDa, its isoelectric point was 4.43, also has the strong hydrophilicity and antigenicity. The derived chemical formula is C1391H2318N412O4878S10, with a negative charge. There is no signal peptide cleavage site and no transmembrane domain in the protein which is an extracellular protein.
Linking the open reading frame of tropomyosin and the pET32a (+) expression vector, and positive clones were picked and sequenced.The correct recombinant plasmid pET32a (+)-Tm was transformed into E. coli BL21 (DE3), and induced expression of tropomyosin with the concentration of 1.0mM/L of IPTG, SDS-PAGE analysis showed that the molecular weight is about 50kDa, which was consisted of a 30.9 kDa protein of the Tm gene and pET-32a(+)(20kDa).The amount of proteinum was to peak at 4 h after IPTG induced. By Western-blotting, the Tm gene was evaluated by immunoblot analysis using recombinant proteins.
For the accurate positioning of tropomyosin's position in the scabies mite,the purified recombinant protein was emulsified with Freund's adjuvant to inject rabbits with vaccine subcutaneous which obtain hyperimmune serum.The EnVision TM +System-HRP(DAB) (DAKO) was used, according to the manufacturer's instructions, for detection of the rabbit antibodies.The tropomyosin-encoding gene of S.scabiei was characterized and the tissue distribution of the proteins encoded by the gene was determined by immunolocalization. Result showed that tropomyosin gene present in the mouthparts, limbs, and internal organs, the epidermis of somato and internal organs, indicating that the gene exists in scabies mite body widespread.
本研究参考GenBank中收录的粉尘螨(D17682)、屋尘螨(AF016278)和痒螨(AM114276)原肌球蛋白(Tropomyosin)基因序列设计引物,对提取的疥螨总RNA进行RT-PCR扩增与克隆,测序结果得到了855bp的目的基因片段。通过对该基因核苷酸序列进行分析,与已报道的粉尘螨、屋尘螨和痒螨原肌球蛋白(Tm)基因的序列同源性分别为87.60%,85.26%和85.03%。预测其表达的重组蛋白大小约为50.90kDa,理论等电点值为pI=4.43,具有很强的亲水性。推导的化学式为C[39] H2318N412O4878S10,带负电荷。该蛋白不存在信号肽切割位点,也不存在跨膜结构域,是一种膜外蛋白。
将目的片段插入pET32a(+)表达载体上,将测序正确的重组质粒转化大肠杆菌BL21(DE3),用浓度为1.0mM/L的IPTG诱导表达了疥螨原肌球蛋白,通过SDS-PAGE分析表明,在相对分子质量约50kDa处有明显的表达条带。进一步优化诱导表达条件,结果表明该重组蛋白在37℃温度下,IPTG浓度为0.4mol/L,诱导4h时基本达到最大表达量。用兔疥螨阳性血清作为一抗进行免疫印迹反应,发现重组表达蛋白可与兔疥螨阳性血清反应,产生蛋白免疫印迹带,说明表达的重组蛋白具有抗原性。
将纯化的重组蛋白与弗氏佐剂混合制备乳剂苗,用其免疫家兔,获得高免血清,并通过免疫组织化学SP技术,对疥螨原肌球蛋白进行组织定位,试验结果表明该蛋白主要在疥螨口器和四肢的肌肉,躯体表皮以及内脏器官表膜中表达。
Sarcoptic mange is a common and highly contagious human zoonotic parasitic disease, highly contagious, which can cause itching and various types of dermatitis, and is a stubborn, contagious skin disease. Over the years, medicines are still the main control of Sarcoptic mange, yet can be used for prevention and treatment of vaccine immunization. The traditional treatment of scabies is usually drug, it is not only ineffective, but also there are drug residues and environmental pollution. Although the development of parasite vaccine is difficult, especially genetic engineering vaccine, but the scabies treatment of genetic engineering vaccine will become the most promising methods with the continuous development of molecular biology, gene library of scabies mites continue to improve.
Here scabies mites from the rabbit were collected, and a pair of primers was designed referencing GenBank. The 855 bp DNA fragment was amplified by polymerase chain reactions,encoding a 285 amino acid residue polypeptide. By blasting the homologous sequence in GenBank databases, the gene which encoded a predicted protein with 87.60%,85.26% and 85.03% identity to the Dermatophagoides farinae allergen Der p10, Dermatophagoides pteronys sinus allergen Der p10 and Psoroptes ovis allergen P. ovis 10 respectively. The molecular mass of polypeptide was 50.90 kDa, its isoelectric point was 4.43, also has the strong hydrophilicity and antigenicity. The derived chemical formula is C1391H2318N412O4878S10, with a negative charge. There is no signal peptide cleavage site and no transmembrane domain in the protein which is an extracellular protein.
Linking the open reading frame of tropomyosin and the pET32a (+) expression vector, and positive clones were picked and sequenced.The correct recombinant plasmid pET32a (+)-Tm was transformed into E. coli BL21 (DE3), and induced expression of tropomyosin with the concentration of 1.0mM/L of IPTG, SDS-PAGE analysis showed that the molecular weight is about 50kDa, which was consisted of a 30.9 kDa protein of the Tm gene and pET-32a(+)(20kDa).The amount of proteinum was to peak at 4 h after IPTG induced. By Western-blotting, the Tm gene was evaluated by immunoblot analysis using recombinant proteins.
For the accurate positioning of tropomyosin's position in the scabies mite,the purified recombinant protein was emulsified with Freund's adjuvant to inject rabbits with vaccine subcutaneous which obtain hyperimmune serum.The EnVision TM +System-HRP(DAB) (DAKO) was used, according to the manufacturer's instructions, for detection of the rabbit antibodies.The tropomyosin-encoding gene of S.scabiei was characterized and the tissue distribution of the proteins encoded by the gene was determined by immunolocalization. Result showed that tropomyosin gene present in the mouthparts, limbs, and internal organs, the epidermis of somato and internal organs, indicating that the gene exists in scabies mite body widespread.
引文
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