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State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094, People’s Republic of China
1 Corresponding author: ninglbau{at}public3.bta.net.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Key Words: human lysozyme • whey • antibacterial activity • transgenic mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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s1-casein promoter. Recombinant human lysozome (rHlys) mRNA was found in the mammary gland tissue of transgenic mice, and the milk of the transgenic mice exhibited antibacterial activity (Maga and Anderson, 1995). However, many studies have shown that the levels of gene expression obtained with genomic DNA-based constructs are generally higher than those obtained with cDNA-based constructs (Brinster et al., 1988; Palmiter et al., 1991), and other research has indicated that the signal peptide is associated with the secretion efficiency of recombinant proteins (Doud et al., 1993; Xiong et al., 2003). Therefore, as a first step toward the ultimate goal of improving the antibacterial properties of bovine milk through creating a mammary gland lysozyme bioreactor, 2 different kinds of transgenic mice containing human lysozyme genomic DNA-based constructs with different signal peptide DNA were respectively established as system models. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Bacteria.
TOP10 was used to clone pBC-hLY and pBC-sighLY expression, and Micrococcus lysodeikticus was used to check lysozyme activity.
Plasmids.
The pBC1 expression vector and the pPCR-XL-TOPO vector were supplied by Invitrogen (Carlsbad, CA), and the bacterial artificial chromosome RP11-1143G9 was from Genentech Inc. (San Francisco, CA).
Molecular Biology Reagents.
The following restriction enzymes were used: NotI and SalI (New England BioLabs Int., Herts, UK), XhoI (Promega, Madison, WI), Csp45I (TaKaRA Bio Inc., Otsu, Shiga, Japan), and HindIII (Huamei Biotechnology Co., Beijing, P. R. China). The following reagents were also used: T4 DNA ligase (New England BioLabs), proteinase K and RNase (Sigma, St. Louis, MO), chicken lysozyme and human lysozyme antibody (Huamei), and -p32-dCTP (Yahui Co., Beijing, P. R. China).
Construction of the rHlys Expression Vectors.
Two rHlys expression vectors, pBC-hLY and pBC-sighLY, were constructed. For construction of the pBC-hLY expression vector, to gain the human lysozyme genomic DNA sequence, we first amplified the genomic region by PCR with the bacterial artificial chromosome RP11-1143G9 as the template. The forward primer was 5' CCC TCG AGA CTC TGA CCT AGC AGT CAA C 3' and the reverse primer was 5' CCG CTC GAG TCT GTT TCT ATC ATT TGG 3'. The reaction conditions were as follows: an initial denaturing step of 94°C for 3 min, followed by 30 cycles of 94°C for 1 min, 60°C for 1 min, and 68°C for 5 min, followed by a final extension step of 68°C for 10 min, and 4°C for 
. The length of the resulting PCR product was 5,652 bp, incorporating the signal peptide DNA sequence and coding sequence (part of exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, and part of exon 4) and a stop codon.
Second, the PCR product was cloned into the pPCR-XL-TOPO vector and was confirmed by sequencing as being the human lysozyme.
Third, the PCR product was cloned into the XhoI restriction site of the pBC1 expression vector (Invitrogen) using conventional molecular cloning techniques, and creation of pBC-hLY was confirmed by sequencing. The pBC-hLY expression vector contained the human lysozyme genomic DNA with its signal peptide DNA, the goat ß-casein promoter, the 3' genomic sequence, exon 1, intron 1, part of exon 2 (before the initial codon), exon 7, intron 7, exon 8, intron 8, and exon 9 of goat ß-casein and 2 copies of chicken ß-globin insulator, as shown in Figure 1A
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). We chose the ß-casein signal peptide because ß-casein is highly expressed in the mammary gland and is secreted efficiently into the milk. A second reason for choosing the ß-casein signal peptide was that goat ß-casein promoter was used to express rHlys in the pBC-sighLY mouse model, although the signal peptide sequences of the different milk proteins are quite similar. ß-Casein is highly conserved between goats and cattle. Therefore, the mammary-specific goat ß-casein promoter and the bovine ß-casein signal peptide should be effective for directing transgene expression in the mammary gland. First, the bovine ß-casein signal peptide DNA sequence was synthesized (indicated by the boldfaced, underlined text) as follows: the forward sequence was 5' TCGAGATGA AGGTCCTCAT CCTTGCCTGC CTGGTGGCTC TGGCCCTTGC AAAGGTCTT 3', and the reverse sequence was 5' C GAAGACCTTT GCAAGGGCCA GAGCCACCAG GCAGGCAAGG ATGAGGA CCT TCATC 3'. The signal peptide DNA was then cloned into the site of pGEM-7zf between XhoI and Csp45 I, and this plasmid was named p7zf-sig.
Second, the human lysozyme genomic DNA was amplified by PCR. The forward primer was 5' ATT CGA AAG GTG TGA GTT GGC CAG AAC TCT G 3', and the reverse primer was 5' TAA GCT TCT CGA GAC CAT CCT GGC TAA CAC G 3'. The PCR conditions were: an initial denaturing step of 94°C for 3 min, followed by 30 cycles of 94°C for 1 min, 60°C for 1 min, and 68°C for 5 min, followed by a final extension step of 68°C for 14 min and 4°C for . The length of the PCR product was 5,255 bp, incorporating the coding sequence (part of exon 1, intron 1, exon 2, intron 2, exon 3, intron 3, and part of exon 4) and the stop codon but not the signal peptide DNA region. The PCR product was cloned into the pPCR-XL-TOPO vector and was confirmed by sequencing. It was then cloned into the site between the HindIII and Csp45 I sites of p7zf-sig, generating the p7zf-sighLY plasmid.
Third, the 5-kb DNA fragment containing the ß-casein signal peptide DNA sequence and the human lysozyme DNA sequence was separated from p7zf-sighLY by digestion with XhoI. The insert was then cloned into the XhoI restriction site of the pBC1 expression vector, producing the pBC-sighLY expression vector, as confirmed by sequencing.
Generation of Transgenic Mice
After the pBC-hLY expression vector and pBC-sighLY expression vector plasmids digested by NotI and SalI, the 25-kb inserts were isolated and recovered by electroelution. To remove any contamination, the products were spot dialyzed against 40 mL of Tris-EDTA (10:0.1) for 30 min (VSWP02500 membrane; Millipore, Milford, MA). Purified DNA was diluted to 2 to 3 ng/µL in TE buffer (10:0.1) and microinjected into the pro-nuclei of fertilized Kunming White x Balb/C eggs. The injected eggs were transplanted into the oviduct of pseudo-pregnant mice.
PCR and Southern Blot Analysis
Chromosomal DNA was isolated from the tails of offspring. Transgenic mice were detected by PCR. The forward primer was 5' TTA TAC ACA CGG CTT TAC 3', and the reverse primer was 5' CAG CAT CAG CGATGT TAT CT 3'. The reaction conditions were an initial denaturing step of 94°C for 5 min, followed by 30 cycles of 94°C for 40 s, 54°C for 40 s, and 72°C for 40 s, followed by a final extension step of 72°C for 7 min and 4°C for 1 h (30 cycles). The length of the PCR product obtained was 637 bp (spanning intron 2 and exon 3).
Ten micrograms of genomic DNA digested by PstI was used to detect transgenic mice. Nontransgenic mouse genomic DNA was used as a negative control and pBC-hLY vector DNA was used as a positive control. An -p32-dCTP-labeled probe (from the former PCR product) was generated and used as the probe in a Southern blot. The positive hybridization signal was a 5-kb fragment.
Three separate positive controls of pBC-hLY or pBC-sighLY vectors containing different copies of the hLY gene were used to estimate the number of copies of the transgene in the transgenic mice by comparing the band density of the positive control with that of the transgenic mice.
Western Blot Analysis
Milk from all viable female founder mice (i.e., I-1, I-3, I-4, II-2, II-4, and II-5) was collected at d 7 of lactation as previously described (Kim et al., 1999). To remove the fat fractions, milk samples were centrifuged at 3,000 x g for 15 min. Liquids from the lower layer were then adjusted to pH 3.8 to 4.6 by 1 M HCl to eliminate the casein fraction. After electrophoresis on a 12.5% SDS-PAGE gel, the proteins were transferred to a nitro-cellulose extra-blotting membrane (Sartorius, Goettingen, Germany). Polyclonal rabbit anti-Hly antibody (Biodesign International, Saco, ME) and horseradish peroxidase-conjugated goat antirabbit-IgG (Sino-American Co., Beijing, China) were used to detect rHlys.
Quantification of rHlys by Radioimmunoassay
For the radioimmunoassay, rHlys (Sigma) was radio-labeled with 125I by the chloramine-T method (Liu et al., 2004). Competition binding was carried out at 4°C overnight in protein binding buffer (0.5% BSA, 0.1% NaN3) using a polyclonal rabbit anti-hLY antibody. Separation of bound and free antibody was achieved by the polyethylene glycol-double antibody method, followed by centrifugation. The radioactivity was then measured using an automatic 
counter.
Assays for Lysozyme Activity
The cell wall of M. lysodeikticus is composed of peptidoglycan polymer, in which the glycosidic ß-(1–4) bonds between N-acetylmuramic acid and N-acetylglucosamine are easily degraded by lysozyme (Hemelt et al., 1979), so M. lysodeikticus is very sensitive to lysozyme. Therefore, the enzymatic activity of lysozyme is routinely determined by monitoring the reduction in turbidity of a suspension of M. lysodeikticus cells at 450 nm (Shugar, 1952; Nilsen et al., 1999; Huang et al., 2002). In this study, 90 µL of an optical density450 = 1.0 M. lysodeikticus cell suspension was made in 1 mM of Tris-Cl buffer, pH 6.24. The cell suspension was equilibrated at room temperature and the reaction was initiated by adding 10 µL of samples containing chicken lysozyme with concentrations of 50, 75, 100, 125, 150, 175, and 200 U/mL. Recombinant human lysozome activity was determined in the kinetic mode for 1 min at 450 nm. The activity of rHlys was calculated relative to a standard curve for native human lysozome, and the rHlys activity of the whey was calculated according to the regression equation deduced from the standard curve.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). The efficiency of integration of the transgene was 7.2%. For pBC-sighLY, 5 transgenic mice (3 females and 2 males) were generated from 63 mice (Figure 2B). The efficiency of integration of the transgene was 7.9%.
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5), which is similar to the results by Liu et al. (2004).
All founder lines except the I-2 founder (which died before maturation) were back-crossed with Kunming White mice to produce the F1 generation. All offspring were screened by PCR. Detection of the transgene in F1 mice indicated that the transgene could be transmitted from the founder transgenic mice to their offspring in a Mendelian manner (Table 1). The transmission rates in some lines, such as lines I-6, II-5, and II-3, were greater than 50%, which may have been due to multiple insertions. The transmission rate was predicted to be 75% if the transgene was inserted in 2 separate nonhomologous chromosomes, as could have been the case for lines I-6 (82.4%) and II-5 (76.9%). The 100% transmission rate in II-3 could have been due to the transgene being inserted into 2 homologous chromosomes, because the F1 offspring of II-3 included male and female mice.
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). The concentration of rHlys was then quantified by radioimmunoassay (Table 1). In pBC-hLY transgenic mice, the level of rHlys in the milk of founder I-1 was 0.154 mg/mL and that of founder I-3 was 0.062 mg/mL, whereas that of founder I-4 was too low to detect. In pBC-sighLY transgenic mice, the levels of rHlys in founders II-4 and II-5 transgenic mice were 1.405 and 0.959 mg/mL, respectively, whereas that of founder II-2 was negligible. The expression levels of rHlys for founders II-4 and II-5 pBC-sighLY transgenic mice were, respectively, 3 and 2 times greater than the concentration of lysozyme in human milk of 0.412 mg/mL. In general, the expression level of rHlys for pBC-sighLY female transgenic mice was much higher than in pBC-hLY transgenic mice.
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We observed that antibacterial activity of the whey from pBC-hLY and pBC-sighLY female transgenic mice was strongly enhanced because of the expression of rHlys. The antibacterial activity of the whey from pBC-hLY female transgenic mice is shown in Table 1. The antibacterial activity of the I-1 transgenic mouse was 480.4 U/mL; of I-3 was 301.6 U/mL; and of I-4 was 37.9 U/mL, whereas that of the nontransgenic mouse and human were 25.9 and 1,224.0 U/mL, respectively. Those of the I-1 and I-3 transgenic mice were, respectively, 18 and 11 times greater than that of the nontransgenic mouse. The antibacterial activity of whey from mouse I-1 was 0.4 times greater than that in humans. Moreover, the antibacterial activity of whey from the pBC-sighLY transgenic mice increased more than that of the pBC-hLY mouse (Table 1). The antibacterial activity levels of the whey for the II-2, II-4, and II-5 transgenic mice were 52.3, 3806.3, and 2,737.5 U/mL, respectively, whereas those of the nontransgenic mouse and human were 25.9 and 1,224.0 U/mL, respectively. The antibacterial activity levels of II-4 and II-5 were 147 and 105 times as high as that in the nontransgenic mouse. Compared with the whey of humans, the antibacterial activity levels of the II-4 and II-5 transgenic mice were 3 and 2 times higher. The antibacterial activity levels of whey from the pBC-sighLY female transgenic mice were generally higher than those of pBC-hLY female transgenic mice, which was consistent with the increased expression levels of rHlys. These results showed that the expression of rHlys may have increased the antibacterial activity of the whey in transgenic mice and confirmed that rHlys is biologically active.
The activity was also calculated in units per milligram (Table 1). The antibacterial activity and concentration of human lysozyme are 1,224.0 and 0.412 mg/mL, respectively, so the specific activity of human lysozyme should be 2,970.9 U/mg. The antibacterial activity in the whey of transgenic mice was mostly due to human lysozyme because mouse lysozyme is virtually inactive (Obita et al., 2003). Therefore, the specific activity of rHlys for the I-1 transgenic mouse was 3,116.8 U/mg. The specific activities of rHlys for the II-4 and II-5 transgenic mice were 2,709.1 and 2,854.0 U/mL, respectively. These results indicate that rHlys had the same specific activity as human lysozyme.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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The transgenic mouse model expressing rHlys was first established by Maga et al. (1994). This model was a cDNA-based construct under the control of the s1-casein promoter (Maga et al., 1994; Maga and Anderson, 1995), in which the expression level of humans varied from 0.25 to 0.71 mg/mL and the specific activity of rHlys was about half that of human lysozyme. Strikingly, the specific activity levels of both pBC-hLY and pBC-sighLY were almost the same as that of human lysozyme. The specific activity of rHlys was similar to that of buffalo milk lysozyme (Parry et al., 1969), was 10 times that of bovine milk lysozyme (White et al., 1988), and was about 3 times that of egg white lysozyme, which is the main commercial lysozyme (Parry et al., 1969). The expression level of pBC-hLY was similar to that reported by Maga et al. (1994), but the expression level of pBC-sighLY was much higher. Although some results have shown that the levels of gene expression obtained with genomic DNA-based constructs are generally higher than those obtained with cDNA-based constructs (Brinster et al., 1988; Palmiter et al., 1991) and that the goat ß-casein promoter can efficiently direct high expression of a foreign gene in the milk of transgenic mice (Roberts et al., 1992; Persuy et al., 1995), no advantage was found in pBC-hLY transgenic mice. Interestingly, the expression of rHlys in pBC-sighLY transgenic mice was remarkably enhanced. This may be for 2 reasons. First, the ß-casein signal peptide may enhance rHlys expression. One possibility to explain this point is that the ß-casein signal peptide is mammary specific. The other possibility is that the ß-casein signal peptide has a favorable amino acid composition, which promotes secretion of rHlys. The idealized core regions of the different signal peptides contain different proportions of Leu and Ala residues, which effectively produce hydrophobicities above and below the threshold level required for efficient secretion (Izard et al., 1995). Doud (1993) observed that if the ratio of Leu and Ala is equal to 6:4, the signal peptide functions most efficiently. In our study, the human lysozyme signal peptide and the bovine ß-casein signal peptide, respectively, were used in the pBC-hLY and pBC-sighLY transgenic mice. The ratio of Leu and Ala for the human lysozyme signal peptide was 5:1, whereas that of bovine ß-casein was 5:4. The data indicate that the signal peptide of bovine ß-casein was more efficient for secreting rHlys. Therefore, our results indicate that different signal peptides affected the expression efficiency of recombinant proteins. Second, the copy number of the transgene in the pBC-hLY and pBC-sighLY transgenic mice may correlate positively with the expression level of rHlys. Based on the data for pBC-hLY and pBC-sighLY (I-1, I-3, II-4, and II-5), a logarithmic regression analysis was performed between the transgene copy number and the lysozyme expression level, and R2 was 0.9856, indicating a strong positive correlation between them. This result supported the view of Reichenstein et al. (2001) that the expression efficiency of a transgene depends on its integration copy numbers, and it is contrary to the conclusion of Thepot et al. (1995) that the copy number of the transgene does not affect its expression efficiency. In addition, we found that not every transgenic founder mouse in both types of transgenic mice expressed rHlys effectively. The chromosomal integration site may still affect the expression pattern of the transgene, although chicken ß-globin insulator was used to prevent position-effect variegation in these 2 systems.
In conclusion, our results show that pBC-hLY and pBC-sighLY transgenic mice can efficiently express the fully active rHlys in the mammary gland, suggesting the feasibility of producing human lysozyme using transgenic farm animals. Compared with the pBC-hLY transgenic mice, the expression levels of rHlys in some lines of pBC-sighLY mice were much higher. This result indicates that the ß-casein signal peptide or copy number of the trangene affects the expression level of rHlys in the mammary gland.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Received for publication November 6, 2005. Accepted for publication February 15, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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-casein-encoding minigene driven by a ß-casein promoter in transgenic mice. Gene 165:291–296.[Medline]This article has been cited by other articles:
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  V. Casaite, S. Bruzyte, V. Bukauskas, A. Setkus, L. A. Morozova-Roche, and R. Meskys Expression and purification of active recombinant equine lysozyme in Escherichia coli Protein Eng. Des. Sel., November 1, 2009; 22(11): 649 - 654. [Abstract] [Full Text] [PDF]
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