人溶菌酶密码子优化及其在牛乳腺细胞中高效表达

doi:10.3864/j.issn.0578-1752.2020.18.015

Abstract

Abstract:

【Objective】The aims of present study were to investigate the codon usage bias of seven milk protein genes in cattle mammary gland, to optimaize human lysozyme gene partially and totally based on codon usage bias of milk protein genes, and to evaluate the codon optimized effect of human lysozyme in multiple cells, so as to provide theoretical basis for increasing recombinant human lysozyme expression and developing new high-effect and safe recombinant human lysozyme. 【Method】The codon usage bias and high/low frequency codons of bovine major milk protein genes were confirmed through bioinformatics analysis by CodonW and EMBOSS software. Human lysozyme gene was partially (22 codons in the translation start region, named LYZop22) and totally (named LYZop) optimized according to the codon usage frequency of bovine major milk protein genes. The human lysozyme-luciferase fusion expression vector (pGL3-LYZcw/op22/op) and overexpression vector (pcDNA-LYZcw/op22/op) were constructed and transfected to cattle mammary epithelial cells (BMEC), cattle fibroblasts (BFFC) and mouse mammary epithelial cells for codon optimization effect evaluation by luciferase, real-time qPCR and western blot methods. 【Result】 The cattle milk protein genes prefer GC end, which the GC3s content was 0.537±0.062. However, the human lysozyme gene prefer AT end, and the GC3s content was 0.407. Moreover, differences in codon usage bias between casein protein genes and whey protein genes were found by cluster analysis. Five high frequency codons (RSCU>1.5) and seven low frequency codons (RSCU<0.5) were found in the bovine milk protein genes.The human lysozyme gene was optimized according to the bovine milk protein codon usage bias and evaluated by multiple cells transfection. The luciferase result showed that, the codon optimization could significantly increase the human lysozyme expression, which were 1.48-fold (P<0.01) and 1.30-fold (P>0.05) increased in BMEC and BFFC of LYZop22, respectively. While, 2.2-fold and 2.44-fold increase in the BMEC and BFFC of LYZop, respectively. Real-time qPCR result showed that, the mRNA levels of human lysozyme were increased in BMEC (2.08-fold, P<0.05) and BFFC (1.5-fold, P>0.05) of LYZop22. While the significantly increase in mRNA level of human lysozyme were found in the BMEC (17.8-fold , P<0.01) and BFFC (22-fold, P<0.01) of LYZop, the totally codon optimized type. In addition, there was a positive correlation between mRNA level and mRNA second structure stability. Western-blot result showed that, the expression of human lysozyme was effectively increased both in partially and totally codon optimized human lysozyme gene in the BMEC. Moreover, the effect on human lysozyme expression in totally codon optimization type was better than partially codon optimization type. 【Conclusion】 The codon usage bias and high/low frequency codons of bovine major milk protein genes were obtained, and the codon optimization of human lysozyme gene according bovine major milk protein gene codon usage bias could significantly improve the mRNA and protein level of human lysozyme, which would lay the foundation for producing recombinant human lysozyme effectively by bioreactor.

Key words: human lysozyme, cattle milk protein genes, codon optimization, luciferase analysis, real-time qPCR, Western-blot

TIAN Yuan,WANG Li,LONG Feng,ZAN LinSen,CHENG Gong. Codon Optimization of Human Lysozyme and High-Efficiency Expression in Bovine Mammary Cells[J].Scientia Agricultura Sinica, 2020, 53(18): 3805-3817.

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Figures/Tables 13

Table 1

Table 2

Table 3

Fig. 1

Table 4

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Table 5

Codon Optimization of human lysozyme gene"

LYZcw	LYZop22	LYZop
1 ATG AAG GCT CTC ATT GTT 19 CTG GGG CTT GTC CTC CTT 37 TCT GTT ACG GTC CAG GGC 55 AAG GTC TTT GAA AGG TGT 73 GAG TTG GCC AGA ACT CTG 91 AAA AGA TTG GGA ATG GAT 109 GGC TAC AGG GGA ATC AGC 127 CTA GCA AAC TGG ATG TGT 145 TTG GCC AAA TGG GAG AGT 163 GGT TAC AAC ACA CGA GCT 181 ACA AAC TAC AAT GCT GGA 199 GAC AGA AGC ACT GAT TAT 217 GGG ATA TTT CAG ATC AAT 235 AGC CGC TAC TGG TGT AAT 253 GAT GGC AAA ACC CCA GGA 271 GCA GTT AAT GCC TGT CAT 289 TTA TCC TGC AGT GCT TTG 307 CTG CAA GAT AAC ATC GCT 325 GAT GCT GTA GCT TGT GCA 343 AAG AGG GTT GTC CGT GAT 361 CCA CAA GGC ATT AGA GCA 379 TGG GTG GCA TGG AGA AAT 397 CGT TGT CAA AAC AGA GAT 415 GTC CGT CAG TAT GTT CAA 433 GGT TGT GGA GTG TAA	1 ATG AAG GCC CTG ATC GTG 19 CTG GGA CTG GTG CTG CTG 37 TCT GTG ACC GTG CAG GGC 55 AAG GTG TTT GAG AGG TGT 73 GAG TTG GCC AGA ACT CTG 91 AAA AGA TTG GGA ATG GAT 109 GGC TAC AGG GGA ATC AGC 127 CTA GCA AAC TGG ATG TGT 145 TTG GCC AAA TGG GAG AGT 163 GGT TAC AAC ACA CGA GCT 181 ACA AAC TAC AAT GCT GGA 199 GAC AGA AGC ACT GAT TAT 217 GGG ATA TTT CAG ATC AAT 235 AGC CGC TAC TGG TGT AAT 253 GAT GGC AAA ACC CCA GGA 271 GCA GTT AAT GCC TGT CAT 289 TTA TCC TGC AGT GCT TTG 307 CTG CAA GAT AAC ATC GCT 325 GAT GCT GTA GCT TGT GCA 343 AAG AGG GTT GTC CGT GAT 361 CCA CAA GGC ATT AGA GCA 379 TGG GTG GCA TGG AGA AAT 397 CGT TGT CAA AAC AGA GAT 415 GTC CGT CAG TAT GTT CAA 433 GGT TGT GGA GTG TAA	1 ATG AAG GCC CTG ATC GTG 19 CTG GGA CTG GTG CTG CTG 37 TCT GTG ACC GTG CAG GGC 55 AAG GTG TTT GAG AGG TGC 73 GAG CTG GCC AGG ACT CTG 91 AAG AGG CTG GGA ATG GAT 109 GGC TAT AGG GGA ATC AGC 127 CTG GCC AAC TGG ATG TGT 145 CTG GCC AAG TGG GAG TCT 163 GGC TAT AAC ACC AGG GCC 181 ACC AAC TAT AAC GCC GGA 199 GAC AGG AGC ACC GAC TAT 217 GGC ATC TTT CAG ATC AAC 235 TCT AGG TAT TGG TGT AAC 253 GAC GGC AAG ACC CCT GGC 271 GCC GTG AAT GCC TGT CAC 289 CTG TCT TGT AGC GCC CTG 307 CTG CAG GAC AAC ATC GCC 325 GAT GCT GTG GCC TGC GCC 343 AAG AGG GTG GTG AGG GAT 361 CCT CAG GGA ATC AGG GCT 379 TGG GTG GCC TGG AGG AAT 397 AGG TGC CAG AAC AGG GAC 415 GTG AGG CAG TAT GTG CAG 433 GGA TGT GGA GTG TAA

Table 5

Fig. 3

Fig. 4

Fig. 5

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Fig. 8

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基因名称 Gene name	GenBank号 GenBank No.	序列长度 Length of CDS
alpha-s1 casein (CSN1S1)	M33123	645 bp
alpha-s2 casein (CSN1S2)	BC114773	669 bp
beta casein (CSN2)	NM_181008	675 bp
kappa casein (CSN3)	BC102120	573 bp
beta-lactoglogbulin (LGB)	NM_173929	537 bp
alpha-lactalbumin (LALBA)	M18780	429 bp
lactotransferrin (LTF)	NM_180998	2127 bp

引物名称 Primer name	基因名称 Gene name	引物序列 Primer sequence	产物长度 Product length	酶切位点 Restriction site
P-pGL3-cw	LYZcw	F：CCATGGCCATGAAGGCTCTCATTG R：CCATGGTTACTCCACATCACTCCACAACCTTGAAC	459bp	NcoI / Nco
P-pGL3-op22	LYZop22	F：CCATGGACCATGAAGGCCCTGATCGTGCTGGGAC TGGTGCTGCTGTCTGTGACCGTGCAGGGCAAGGT GTTTGAGAGGTGTGAGTTGGCCAGAACTCTG R：CCATGGTCACTCCACAACCTTGAAC	459bp	NcoI / NcoI
——	LYZop	全基因合成 Whole gene synthesis	459 bp	NcoI / NcoI
P-pcDNA-cw	LYZcw	F：AAGCTTCCATGAAGGCTCTCATTG R：CTCGAGCTCCACAACCTTGAAC	456 bp	HindIII / XhoI
P-pcDNA-op22	LYZop22	F：CCATGGACCATGAAGGCCCTGATCG R：CTCGAGCTCCACAACCTTGAAC	456bp	HindIII / XhoI
P-pcDNA-op	LYZop	F：CCATGGACCATGAAGGCCCTGATCG R：CTCGAG CACTCCACATCCCTGCACAT	456 bp	HindIII / XhoI

Bos taurus	A3s	T3s	C3s	G3s	GC3s	GC	CAI	ENc
αs1酪蛋白alpha-s1 casein	0.311	0.461	0.290	0.258	0.408	0.445	0.227	52.300
αs2酪蛋白alpha-s2 casein	0.253	0.453	0.340	0.335	0.484	0.417	0.317	49.970
β酪蛋白beta casein	0.201	0.421	0.342	0.298	0.505	0.512	0.234	48.750
κ-酪蛋白 kappa casein	0.399	0.412	0.243	0.194	0.341	0.430	0.240	58.250
乳球蛋白lactoglogbulin	0.079	0.147	0.574	0.519	0.825	0.586	0.349	36.270
α乳清白蛋白alpha-lactalbumin	0.283	0.349	0.395	0.337	0.526	0.441	0.281	55.510
乳铁蛋白lactotransferrin	0.170	0.242	0.410	0.449	0.671	0.561	0.262	47.650
Mean ± SEM	0.242±0.039	0.355±0.045	0.371±0.040	0.341±0.042	0.537±0.062	0.485±0.026	0.273±0.017	49.814±2.668
人溶菌酶 human lysozyme	0.3143	0.4032	0.2742	0.2323	0.407	0.473	0.193	50.270

密码子 Codon	氨基酸 Amino acid	频率/1000 Frequency/1000	个数 Number	同义密码子相对使用度RSCU	密码子 Codon	氨基酸 Amino acid	频率/1000 Frequency/1000	个数 Number	同义密码子相对使用度RSCU
GCA	A	13.793	26	0.73	AAT	N	19.629	37	0.96
GCC	A	32.891	62	1.73	CCA	P	17.507	33	1.06
GCG	A	8.488	16	0.45	CCC	P	19.629	37	1.19
GCT	A	20.69	39	1.09	CCG	P	4.244	8	0.26
TGC	C	14.854	28	0.98	CCT	P	24.403	46	1.48
TGT	C	15.385	29	1.02	CAA	Q	16.976	32	0.57
GAC	D	22.281	42	1.09	CAG	Q	42.44	80	1.43
GAT	D	18.568	35	0.91	AGA	R	5.836	11	1.06
GAA	E	33.952	64	0.90	AGG	R	12.732	24	2.32
GAG	E	41.91	79	1.10	CGA	R	2.122	4	0.39
TTC	F	21.22	40	1.13	CGC	R	3.183	6	0.58
TTT	F	16.446	31	0.87	CGG	R	6.897	13	1.26
GGA	G	11.671	22	1.07	CGT	R	2.122	4	0.39
GGC	G	15.385	29	1.41	AGC	S	14.324	27	1.30
GGG	G	9.019	17	0.83	AGT	S	13.263	25	1.20
GGT	G	7.427	14	0.68	TCA	S	7.427	14	0.67
CAC	H	11.141	21	1.35	TCC	S	14.854	28	1.34
CAT	H	5.305	10	0.65	TCG	S	2.122	4	0.19
ATA	I	2.653	5	0.18	TCT	S	14.324	27	1.30
ATC	I	24.403	46	1.64	ACA	T	11.141	21	0.84
ATT	I	17.507	33	1.18	ACC	T	18.037	34	1.36
AAA	K	33.952	64	0.88	ACG	T	6.366	12	0.48
AAG	K	42.971	81	1.12	ACT	T	17.507	33	1.32
CTA	L	4.244	8	0.25	GTA	V	6.897	13	0.41
CTC	L	19.629	37	1.14	GTC	V	15.915	30	0.95
CTG	L	46.684	88	2.71	GTG	V	28.117	53	1.68
CTT	L	18.037	34	1.05	GTT	V	15.915	30	0.95
TTA	L	2.122	4	0.12	TAC	Y	15.385	29	0.91
TTG	L	12.732	24	0.74	TAT	Y	18.568	35	1.09
AAC	N	21.22	40	1.04

Codon Optimization of Human Lysozyme and High-Efficiency Expression in Bovine Mammary Cells

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