Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (10): 1830-1838.doi: 10.3864/j.issn.0578-1752.2019.10.015

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles    

Expression, Purification and Localization Analysis of Polar Tube Protein 2 (NbPTP2) from Nosema bombycis

YI Min1,2,LÜ Qing1,LIU KeKe1,WANG LiJun1,WU YuJiao1,ZHOU ZeYang1,LONG MengXian1,2()   

  1. 1 State Key Laboratory of Silkworm Genome Biology, Southwest University/Chongqing Key Laboratory of Microsporidia Infection and Prevention, Chongqing 400715
    2 College of Biotechnology, Southwest University, Chongqing 400715
  • Received:2019-01-11 Accepted:2019-03-15 Online:2019-05-16 Published:2019-05-23
  • Contact: MengXian LONG E-mail:longmx@swu.edu.cn

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Abstract:

【Objective】Microsporidia are eukaryotic intracellular obligate parasites that infect almost all organisms, including human. As a special infection organ, the polar tube is mainly composed of polar tube proteins. The polar tube protein plays an important role in microsporidia invasion host and maintaining the structure of polar tube. The objective of this study is to clone and express Nosema bombycis polar tube protein 2 (NbPTP2), analyze its localization characteristics in mature spores, and to lay a foundation for further study the function of polar tube proteins.【Method】NbPTP2 was amplified from N. bombycis genome. The amino acid composition, theoretical molecular weight and predicted isoelectric point of NbPTP2 were analyzed by Expasy online software. SignalP 4.1 and TMHMM Server V. 2.0 were used to predict the signal peptide and transmembrane domain of NbPTP2. The phosphorylation site of NbPTP2 was analyzed by NetPhos 3.1 Server. The phylogenetic tree of NbPTP2 from different microsporidia species was constructed by MEGA 7.0. NbPTP2 was amplified from N. bombycis genome, then ligated with prokaryotic expression vector pET32a (+). The correctly sequenced recombinant plasmid was transformed into Escherichia coli Rosetta, and protein expression was heterologous induced by IPTG. The polyclonal antibody of NbPTP2 was prepared by immunizing New Zealand rabbits with the fusion protein by affinity chromatography purification. The expression of NbPTP2 in mature spores was detected by Western blot. Indirect immunofluorescence assay (IFA) was used to analyze the localization characteristics of NbPTP2 in mature spores of microsporidia. 【Result】 The NbPTP2 with a length of 834 bp was successfully cloned. The protein encodes 278 amino acid residues with a theoretical molecular weight of 30.9 kD, and isoelectric point of 9.39. Moreover, it was predicted to have a N-terminal signal peptide and potential phosphoric acid sites, but no transmembrane domain. The phylogenetic tree analysis result showed that NbPTP2 from N. bombycis was closely related to NaPTP2 from N. apis and NcPTP2 from N. ceranae. Western blot result showed that NbPTP2 was expressed in mature spores of N. bombycis and its molecular weight was about 39 kD. The localization analysis result of IFA indicated that NbPTP2 could locate on the whole polar tube of N. bombycis, and it was confirmed that NbPTP2 was a polar tube protein. 【Conclusion】 The relationship between NbPTP2 and polar tube protein 2 from other microsporidia was clarified. NbPTP2 was expressed in N. bombycis and could be localized on the whole polar tube after germination. These results can provide a basis for polar tube structure analysis and polar tube protein function research.

Key words: silkworm (Bombyx mori), Nosema bombycis, polar tube protein 2, prokaryotic expression, localization analysis

Fig. 1

Phylogenetic tree of PTP2 from different genus and species microsporidia"

Fig. 2

PCR and enzyme digestion analysis of pET32a(+)- NbPTP2 recombinant plasmid"

Fig. 3

SDS-PAGE analysis of expression and purification of pET32a(+)-NbPTP2 recombinant protein"

Table 1

LC-MS/MS analysis of recombinant protein"

蛋白
Protein		 等电点/相对分子质量
PI/MW (kD)		 唯一肽段数
UniquePepCount		 肽段覆盖率
CoverPercent		 肽信息Peptide information
起始位点
Start site		 结束位点
End site		 序列
Sequence
Polar tube protein 2
（EOB15197.1）		 9.39/30.9 23 75.09% 1 8 MFLSLNRK
8 21 KLFTVASLLTVIRT
20 38 RTMASVAPPQGGIMVPNRA
37 51 RAQQQMLPAVVDPRK
51 63 KAVCNIQYINGKK
63 78 KIIPANNNNPAECQRD
77 83 RDLIERA
86 97 RQAQAIETAARA
96 106 RAVQTIEHIKQ
96 110 RAVQTIEHIKQEPKC
117 124 RETQCIKT
125 132 KLLNELKN
125 145 KLLNELKNEPEYTVTGEENKV
131 145 KNEPEYTVTGEENKV
144 150 KVNVFKK
144 151 KVNVFKKG
152 167 KKIMLAANIQHHFIKV
153 167 KIMLAANIQHHFIKV
166 180 KVEKPPSEQFQIVKK
180 198 KAKEAYVFNAIGEVLSTKQ
201 213 KRPAPQCICPGKS
212 229 KSTPQTAEGTPLINECKQ
228 234 KQAECKN

Fig. 4

Western blot analysis of NbPTP2 expressed in total protein from N. bombycis"

Fig. 5

Indirect immunofluorescence assay analysis of NbPTP2 localization in N. bombycis"

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