Biosynthesis of Ctenopharyngodon idella C-type Lysozyme Based on Recombinant Pichia pastoris and Its Antibacterial Activity

doi:10.13560/j.cnki.biotech.bull.1985.2021-0256

Abstract

Abstract:

C-type（Chicken-type）lysozyme,as an important protein immune factor in the endogenous immune system of grass carp（Ctenopharyngodon idella）,can play an important role in the resistance of grass carp to pathogenic microorganisms,it is also a good choice for the development of green feed additives or biological antimicrobial agents. In the present study,the cDNA encoding grass carp C-type lysozyme（CilyC）was cloned by reverse transcription PCR（RT-PCR）. Subsequently,two times of PCR were performed to add various necessary sites to its 5' and 3' ends. The CilyC was connected to the expression vector “pPICZαA” and then transferred into the engineering strain Pichia pastoris X-33;thus,the recombinant P. pastoris strain X-33/pPICZαA-CilyC was constructed. The recombinant strains with multiple copies of gene inserts were screened by using a medium containing high concentration of zeocin. Protein expression conditions for the recombinant strains were optimized and screened. The expression products were purified by nickel ion affinity chromatography,the purified protein was subjected to Western blot analysis and LC-MS/MS identification. In addition,the antibacterial activity of the recombinant strain expression products was detected by plate coating and MIC（minimum inhibitory concentration）method. The results showed that the X-33/pPICZαA-CilyC produced 13.7 mg/L recombinant protein under the optimal fermentation conditions（29^oC,250 r/min,96 h,and 1% methanol）. The recombinant protein was structurally identified as an expected CilyC protein with a molecular weight of 14.5 kD. The results of bacteriostatic tests showed that the recombinant CilyC had obvious biological activities against Gram-positive Staphylococcus aureus,Bacillus subtilis,Listeria monocytogenes and Bacillus cereus,as well as Gram-negative Pseudomonas aeruginosa and Salmonella. The recombinant P. pastoris strain “X-33/pPICZαA-CilyC” constructed in this study may effectively synthesize the grass carp C-type lysozyme,which lays a good foundation for the large-scale preparation of fish-derived C-type lysozyme.

Key words: Ctenopharyngodon idella, C-type lysozyme, recombinant Pichia pastoris, antibacterial activity

YANG Yue, TAO Yan, XIE Jing, QIAN Yun-fang. Biosynthesis of Ctenopharyngodon idella C-type Lysozyme Based on Recombinant Pichia pastoris and Its Antibacterial Activity[J]. Biotechnology Bulletin, 2021, 37(12): 169-179.

Figures/Tables 11

Table 1 Primer sequences

Primer name	Primer sequence（5'-3'）	Size/bp
CF	TCTTCAGATAGCAGAAATTGACAGT	25
CR	TAGGTAATTAAAGATCCCTCAGGCAA	26
CP1	CATCATCATCATCATCATCGCACAATGGG	29
CP2	TCTAGATTATCAGTGCGATTCGCA	24
CP3	CTCGAGAAAAGACATCATCATCATCATCATC	31
3'AOX1	GCAAATGGCATTCTGACATCC	21
5'AOX1	GACTGGTTCCAATTGACAAGC	21

Fig. 1 Nucleotide and deduced amino acid sequences of the target gene CilyC Bold letters refer to 6×His;italics refer to Kex2 signal peptidase cleavage site. Underlines refer to Xho I and Xba I restriction endonuclease sites. Shaded letters refer to conserved cysteine residues. Boxes refer to active site residues

Fig.2 Three-dimensional structure of the CilyC protein Different numbers of Cys refer to cysteine residues. Red,green,yellow and blue refer to 4 pairs of disulfide bonds. Orange and pink refer to residues of the active site

Fig. 3 Identification of recombinant expression vector pPICZαA-CilyC by colony PCR（A）and restriction endonuclease digestion（B） M:DNA ladder. 1-9:Colony PCR products. 10:Products digested by restriction endonucleases

Fig. 4 PCR identification for the genome DNA of recom-binant P. pastoris strain M:DNA ladder. 1-8:PCR products

Fig. 5 Tricine-SDS-PAGE analysis for pre-expressed products of the recombinant strains M:Ultra-low molecular weight protein marker. 0:Expression product of the X-33/pPICZαA. 1-3:Expression products of X-33/pPICZαA-CilyC

Fig. 6 Effects of methanol concentration（A）and expression time（B）on the expression products of the recombinant strain There is no significant difference among the same letter items,and there is a significant difference among different letter items（P <0.05）. Data are in format of mean ±SD,n=3. M:Ultra-low molecular weight protein marker

Fig. 7 Tricine-SDS-PAGE（A）and Western blot（B）analysis for the purified protein M:Rainbow pre-stained protein marker. 1:Expression product of the X-33/pPICZαA. 2:Purified protein

Fig. 8 LC-MS/MS analysis for the purified protein Capital letters refer to amino acid sequences of the peptide fragments

Fig. 9 Antibacterial activities of the recombinant strain culture supernatant against Gram-positive and Gram-negative bacteria A:Bacteria treated with the culture supernatant of X-33/pPICZαA. B:Bbacteria treated with the culture supernatant of X-33/pPICZαA-CilyC

Table 2 Minimum inhibitory concentration（MIC）of the recombinant CilyC against different test bacteria

受试菌株 Test strain	MIC/（µg·mL^-1）
革兰氏阳性细菌Gram-positive
金黄色葡萄球菌Staphylococcus aureus	60
枯草芽孢杆菌Bacillus subtilis	80
单增李斯特菌Listeria monocytogenes	80
蜡样芽孢杆菌Bacillus cereus	80
革兰氏阴性细菌Gram-negative
沙门氏菌Salmonella	80
铜绿假单胞菌Pseudomonas aeruginosa	120

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