Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (17): 3059-3068.doi: 10.3864/j.issn.0578-1752.2019.17.013

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

The Expression Pattern and Ligand Binding Ability of Hc-FAR-4 Protein of Haemonchus contortus

WEI HaiDian,CHEN XueQiu,HUANG Yan,SHI HengZhi,ZHOU JingRu,WU Fei,DU AiFang,YANG Yi()   

  1. College of Animal Science, Zhejiang University/Key Laboratory of Animal Preventive Medicine of Zhejiang Province, Hangzhou 310058
  • Received:2018-12-06 Accepted:2019-02-26 Online:2019-09-01 Published:2019-09-10
  • Contact: Yi YANG E-mail:yangyi0607@zju.edu.cn

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Abstract:

【Objective】 Haemonchus contortus is a parasite mainly settled in the abomasum mucosa of small ruminants. The disease caused by H. contortus is a national epidemic in China. In this paper, the expression pattern and ligand binding ability of Hc-FAR-4 were studied to understand its role in the growth, development and reproduction of H. contortus.【Method】 The prokaryotic expression vector pET-30a-Hc-far-4 was constructed, which was identified by PCR and enzyme digestion identification, and then, the recombinant plasmid was transformed into E. coli BL21. The recombinant protein was induced by 0.1 mmol·L -1 IPTG (isopropyl-β-d-thiogalactoside). The recombinant protein rHc-FAR-4 was collected and was identified by Western Blot. Fluorescence analysis method, and then, which was used to study the binding ability of Hc-FAR-4 protein with DAUDA, retinol, and oleic acid. The IHF experiment was based on the fluorescent material retinol and fatty acid analogues DAUDA in polar or nonpolar solution, and their excitation spectra characteristics would change when they were excitated by a certain wavelength of exciting light. Using fluorescence analysis method, we could estimate whether rHc-FAR-4 protein contained the ability to bind with DAUDA and retinol by the change of the excitated spectra. When non-fluorescent oleic acid was added into the system, it would compete with DAUDA and retinol to bind with the bind site of rHc-FAR-4 protein, and which would make excitated spectral change, too. According to the changes, we could indirectly judge whether rHc-FAR-4 protein could bind with non-fluorescent oleic acid. At the same time, polyclonal antibodies were prepared by using rHc-FAR-4 protein to immunize mice, and the antibody titer of the immunized mice was tested by ELISA. The serum of the immunized mice would be collected if the antibody titer was appropriate. The collected serum was used in the immunofluorescence (IHF) experiment to explore the expression site of Hc-FAR-4 protein to speculate its function in H. contortus. The IHF experiment process was as follow: The H. contortus was embedded in paraffin; the paraffin was cut into slices; Antigen was repaired; the slices was incubated by 3% BSA at 4℃ for one night; slices was incubated by anti-Hc- FAR-4 mouse antibody and Alexa Fluor? 488 nm goat anti-mouse IgG antibody for 1 h, and which was done as primary and secondary antibody, respectively; after staining by DAPI, the slices were observed by confocal microscopy. What ’s more, the qPCR technology was used to analyze the expression characteristics of Hc-far-4 in H. contortus.【Result】 The target gene Hc-far-4 was successfully cloned. The recombinant plasmid pET-30a-Hc-far-4 was successfully expressed in E. coli BL21. The recombinant plasmid pET-30a-Hc-far-4 was successfully expressed in E. coli BL21, and the expression level of rHc-FAR-4 protein reached peak after induction for 8h. The result of ELISA showed that the titer of mouse polyclonal antibody was 1:1 024 000—1:2 048 000, which could be used in the following experiments. Western Blot result showed that the rHc-FAR-4 protein contained His tag and the band size was 25 kD, which was consistent with the prediction. The mouse polyclonal antibody was identified by Western Blot and could bind to natural Hc-FAR-4 protein, indicating that the antibody could be used in IHF experiment. The results of ligand binding experiments of rHc-FAR-4 showed that rHc-FAR-4 could bind to fatty acids and retinol. The qPCR analysis showed that Hc-far-4 reached the highest transcription level in the fourth stage larvae. Immunohistofluorescence assay showed that Hc-FAR-4 was mainly expressed in the intestinal wall and gland of H. contortus. To sum up, it could be speculated that Hc-FAR-4 protein might be involved in the transport of fatty acids and retinol, and provided nutrients for H. contortus to ensure its normal growth, development and reproduction; what ,s more, Hc-FAR-4 protein maybe also involved in the process of modifying host tissues to enable the parasite to escape the immunity of host.【Conclusion】 rHc-FAR-4 could bind to DAUDA and retinol, and which was mainly expressed in the intestinal wall, cuticle and gonad. The transcription level of Hc-far-4 reached the peak in the fourth stage larvae.

Key words: Haemonchus contortus, Hc-far-4, expression pattern, ligand binding capacity, fatty acids

Fig. 1

Cloning of Hc-far-4 gene fragment and identification of pET-30a-Hc-far-4 by digestion A. Amplification result of Hc-far-4; B. Enzyme digestion identification result of pET-30a-Hc-far-4. M: DL250 DNA marker; A-1: PCR product of Hc-far-4; B-1: Enzyme digestion product of pET-30a-Hc-far-4 plasmid"

Fig. 2

rHc-FAR-4 protein expressed in E. coli BL21 M: Protein marker; 1: pET-30a empty plasmid; 2: pET-30a-Hc-far-4 were inducted for 0 h; 3: pET-30a-Hc-far-4 were inducted for 4 h; 4: pET- 30a-Hc-far-4 were inducted for 6 h; 5: pET-30a-Hc-far-4 were inducted for 8 h"

Fig. 3

SDS-PAGE analysis of purified protein and its Western blot analysis A. SDS-PAGE analysis result of rHc-FAR-4 protein; B. Western Blot identification result of rHc-FAR-4 protein. M: Protein molecular weight marker; A-1, B-1: rHc-FAR-4 protein"

Fig. 4

The change of fluorescence emission spectra induced by the addition of rHc-FAR-4 proteins into ligand solution A: DAUDA binding experiment of rHc-FAR-4 protein; B: Retinol binding experiment of rHc-FAR-4 protein"

Fig. 5

Western Blot analysis of the specificity of mouse polyclonal antibody A: Anti-rHc-FAR-4 mouse polyclonal antibody do as primary antibody; B: Negative serum of mouse do as primary antibody. M: protein marker; A-1, B-1: Whole worm protein of H. contortus"

Fig. 6

Transcriptional levels of Hc-far-4 at different developmental stages of H. contortus"

Fig. 7

Expression location of Hc-FAR-4 in L4F of H. contortus A: Negative control; B: transection of H. contortus; C: longitudinal cutting of H. contortus. a: Intestinal wall; b: Gonads; C: cuticle; Column 1: DAPI; Column 2: FITC; Column 3: DIC; Column 4: Merge"

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