Construction and Identification of Intestine-specific Expression Vector of Xylanase Gene (XynB)

Sheng GAO Yulong WANG Zhe CHEN Hui KE

Abstract The Aspergillus niger XynB gene and core promoter region of porcine RELMβ gene were cloned into pcDNA3.1（-）， and an intestine-specific expression vector pcDNA3.1-RELMβ-XynB-Myc-GFP carrying green fluorescence and Myc double tags was constructed. The vector was transfected into human colon cancer cells （HT29） and human liver cancer cells （Bel7402） using liposomes. Fluorescence microscopy revealed that the vector could specifically express green fluorescent protein （GFP） in HT29 cells. RT-PCR and Western Blot were performed on the HT29 cells transfected with the expression vector， and the results showed that the XynB gene was normally transcribed in HT29 cells， and the target protein expression was detected in the cells.

Key words Xylanase; Specific expression vector; Resistin-like β gene; WB detection; RT-PCR detection

Xylan is a hemicellulose polysaccharide whose content is second only to cellulose. As the main anti-nutritional factor in corn-soybean meal type diets， it cannot be independently digested by endogenous digestive enzyme in monogastric animals[1]. Xylanase （XynB） can destroy the macromolecular structure of xylan and is the key enzyme for hydrolyzing xylan. The xylanase from Aspergillus niger has good acid resistance， and the temperature and pH value of the gastrointestinal tract of livestock and poultry have no obvious effect on the xylanase activity. xylanase was widely used as an additive in livestock and aquatic feed production[2]. Considering the degradation efficiency of exogenous xylanase and the widespread existence of xylan， the expression of xylanase in the intestinal tract of animals using transgenic technology provides the possibility for the complete degradation of xylan in feed.

Resistin-like molecule β （RELMβ） is an important candidate gene related to intestinal immune protection， which is specifically and highly expressed in the proximal and distal colon[3]. In this study， The A. niger XynB gene and core promoter region of porcine RELMβ gene were cloned into pcDNA3.1（-） vector， and an intestine-specific expression vector carrying green fluorescence and Myc double tags was constructed， aiming to provide materials for the endogenous secretion of xylanase， and also provide a preliminary basis for further improving feed utilization and reducing livestock manure pollution.

Materials and Methods

Materials

Strains and Plasmid

pcDNA3.1（-） plasmid， purchased from Invitrogen; pMD18T plasmid， purchased from Takara; pRSETA-XynB plasmid， constructed and endowed by the research group of Professor Wu， College of Animal Science， South China Agricultural University; pEGFP-C3 plasmid and pMD18-RELMβ （-574-+215） plasmid， preserved in our laboratory; human colon adenocarcinoma cell line HT29 and human liver cancer cell line Bel7402， purchased from Nanjing KeyGen Biotech. Co. Ltd.

Main reagents and tool enzymes

Restriction enzymes Nhe I， Xho I， Kpn I， Prime STAR high-fidelity DNA polymerase， T4 DNA ligase， Premix Taq （LaTaqversion2.0） enzyme， DH5α competent cells， all purchased from Takara; Endo-free Plasmid Midi Kit， gel recovery kit， and rapid ligation kit （LigaFastTM Rapid DNA Ligation System）， purchased from Promega.

Transfection kit LipofectamineTM LTX， purchased from Invitrogen; trypsin， PBS， DMEM （high glucose）， and fetal bovine serum （FBS）， Gibco products; anti-Myc mouse primary antibody， horseradish peroxidase-coupled goat anti-mouse secondary antibody and Pro-light HRP chemiluminescence detection reagent， purchased from Tiangen Biotech （Beijing） Co. Ltd.; WesternBlot related reagents， purchased from Sangon Biotech （Shanghai） Co. Ltd.; developer and fixer， purchased from Nanjing Sitaile Company.

Methods

Construction of eukaryotic expression vector pcDNA3.1-XynB-Myc

With the prokaryotic vector pRSETA-XynB as a template， according to the XynB gene sequence， the PCR primers P1F/P1R （Table 1） were designed. The 5′ end primer P1F had the Xho I restriction site （CTCGAG） and its protective base， and the 5′ end primer P1R was introduced with the Myc-Tag sequence and had the Kpn I restriction site （GGTACC） and its protective base. The PCR product was connected with the pMD18T vector to construct the cloning plasmid pMD18-XynB-Myc， which was transformed into Escherichia coli DH5α for blue-white selection. The positive plasmid was identified by double restriction digestion and sequencing. The pcDNA3.1（-） vector and pMD18-XynB-Myc recombinant plasmid were digested with restriction enzymes Xho I and Kpn I， respectively， and the vector pcDNA3.1（-） and the target fragment XynB-Myc were purified and recovered. The purified and recovered XynB-Myc target fragment was ligated to the linearized pcDNA3.1（-） vector using T4 ligase to construct the XynB eukaryotic expression vector pcDNA3.1-XynB-Myc， and the ligation product was transformed into DH5α competent cells. After blue-white selection， positive transformants were selected to expand in LB liquid medium， and then a small amount of plasmids were extracted and identified by enzyme digestion.

Construction of pcDNA3.1-XynB-Myc-GFP

According to the plasmid pcDNA3.1-XynB-Myc， PCR primers P1F/P2R was designed （Table 1）. The 5′ end of the P1F primer had the restriction site Xho I， and the 5′ end sequence of the P2R primer was complementary to the 5′ end of the GFP sequence. Samples were denatured at 98 ℃ for 5 min followed by 30 cycles under the following conditions： denaturation for 30 s at 98 ℃， annealing for 30 s at 58C and extension for 30 s at 72 ℃， and completed with 72 ℃ for 10 min， and the size of the amplified XynB-Myc fragment was 682 bp. With the pEGFP-C3 sequence as a template， the PCR primers P3F/P3R were designed （Table 1）. The 5′ end of P3F was consistent with the 3′ end of the XynB-Myc sequence， and the 5′ end of the primer P3R had a restriction site Kpn I. The amplification program had 30 cycles as following： 98 ℃ for 5 min， 98 ℃ for 30 s， 62 ℃ for 30 s and 72 ℃ for 30 s， and 72 ℃ for 10 min， and the size of the amplified fragment was 768 bp.

Use the above amplified products XynB-Myc fragment and GFP fragment as templates， the two components were fused by overlap PCR using primers P1F and P3R to obtain the fusion fragment XynB-Myc-GFP （1 398 bp）. The amplification program was started with 98 ℃ for 4 min， followed by 30 cycles of 98 ℃ for 10 s， 52 ℃ for 10 s， 72 ℃ for 130 s， and completed with 72 ℃ for 10 min. The size of the amplified fragment was 1 398 bp.

After purification of the fusion fragment XynB-Myc-GFP， it was ligated with pMD18T using T4 ligase to obtain the intermediate vector pMD18TXynB-Myc-GFP， which was identified by double enzyme digestion. Double enzyme digestion was performed on pMD18T-XynB-Myc-GFP with Xho I and Kpn I， and the XynB-Myc-GFP fragment was recovered and cloned to the corresponding restriction site of pcDNA3.1（-） to obtain the recombinant vector pcDNA3.1-XynB- Myc-GFP， which was then identified by double enzyme digestion.

Construction of intestine-specific expression vector guided by porcine RELMβ gene promoter

Double enzyme digestion was performed on pMD18-RELMβ （-574-+215） and pcDNA3.1-XynB-Myc-GFP with Nhe I and Xho， followed by gel recovery of the target fragment. The RELMβ core promoter fragment was cloned into the corresponding restriction site of pcDNA3.1-XynB-Myc-GFP to obtain the recombinant vector pcDNA3.1-RELMβ-XynB-Myc-GFP， which was identified by restriction digestion.

Specific expression of XynB in HT29 cells

HT29 cells were inoculated in culture plate wells， and pcDNA3.1-RELMβ-XynB-Myc-GFP recombinant vector was transfected using liposomes. After 12 h of transfection， the expression of green fluorescent protein was observed and photographed using an inverted microscope.

Identification of XynB gene expression in HT29 cells

The HT29 cell line stably transfected with the recombinant expression vector was resuscitated， and the total cell protein after 48 h of culture was used for Western-blot test. The Myc antibody was selected to detect the expression of the target protein XynB in the cells. RNA was extracted from the transgenic HT29 cell line cultured for 4 d， normally cultured HT29 cells of the same generation， and HT29 cells transfected with empty vector. After DNase treatment， the transcription results were detected by RT-PCR. Primers were designed to amplify partial sequence of the XynB-GFP gene fragment， and primers for amplifying β-actin locus covering exon and intron regions were designed to eliminate template DNA contamination （Table 1）.

Results and Analysis

Construction of eukaryotic expression vector pcDNA3.1-XynB-Myc

With the prokaryotic vector pRSETA-XynB as a template， the target gene fragment XynB-Myc was amplified by PCR， with a size of 678 bp （Fig. 1-A）. Double enzyme digestion showed that XynB-Myc was successfully ligated into the pMD18T vector （Fig. 1-B）. The recombinant vector pcDNA3.1-XynB-Myc was subjected to double enzyme digestion， and two bands were obtained， which were in line with expectations （Fig. 1-C）， indicating that XynB-Myc was successfully inserted into the expression vector pcDNA3.1.

Construction of vector pcDNA3.1-XynB-Myc-GFP

The size of the amplified XynB-Myc fragment was 682 bp. With pEGFP-C3 as the template， the size of the amplified GFP fragment was 768 bp. With the XynB-Myc fragment and GFP fragment were used as templates， overlap PCR was used to obtain the XynB-Myc-GFP fusion fragment， which was 1 398 bp in size， and the electrophoresis detection showed that the digested product was in line with the expected size （Fig. 2-A）. Double restriction digestion showed that XynB-Myc-GFP was successfully ligated into the pMD18T vector （Fig. 2-B）. The constructed pcDNA3.1-XynB-Myc-GFP expression vector was digested with double restriction enzymes Xho I and Kpn I， and the target ligated fragment and empty plasmid fragment with the expected sizes were observed through gel electrophoresis （Fig. 2-C）， indicating the XynBMyc-GFP fragment was successfully cloned into pcDNA3.1 vector.

Construction of intestine-specific expression vector pcDNA3.1-RELMβ-XynB-Myc-GFP

In the previous study， the expression characteristics and promoter activity of porcine RELMβ gene was done， and the core promoter region （-574-+215） was selected as the intestine-specific promoter and cloned into the expression vector. The constructed pcDNA3.1-RELMβ-XynBMyc-GFP vector was detected by gel electrophoresis after double enzyme digestion. The target ligated fragment and the empty plasmid fragment with a size of about 2 000 bp were observed， indicating that the RELMβ promoter fragment was successfully inserted into pcDNA3.1-XynB-Myc-GFP vector （Fig. 3）.

The specific expression of XynB in HT29 cells

According to the liposome method， the recombinant expression vector pcDNA3.1-RELMβ-XynB-Myc-GFP was transfected into HT29 and Bel7402 cells， which were then cultured in vitro. After 12 h of culture， the fusion expression of the GFP could be observed using fluorescence microscope in HT29 cell， while there was no fluorescence in the Bel7402 cells （Fig. 4）. The results show that the constructed intestine-specific expression vector could guide the specific expression of XynB gene in intestinal cells.

Identification of gene XynB in HT29 cells

It can be seen from Fig. 5 that the XynB gene-transformed HT29 cell protein contained the target protein， which was about 50 ku in size. The target protein is not detected in the negative control group.

RNA was extracted as a template， and RT-PCR detection was performed. It can be seen from Fig. 6 that the XynB enzyme mRNA sequence was normally transcribed and synthesized in the transgenic HT29 cells. The amplification of β-actin gene with cross-intron primers showed no intron fragment amplification， eliminating the possibility of DNA contamination.

Conclusions and Discussion

There are a large number of natural promoters and each has its own characteristics. When conducting studies on in-vitro expression of gene recombinants， researchers usually introduce specific promoters to achieve tissue-specific expression of target genes. β-lactoglobulin （BLG） is the most important whey protein in ruminants， and transgenic animals such as the fusion α-antitrypsin， calcitonin and human serum albumin constructed using the BLG gene promoter and 5′ end regulatory sequences can express target gene in the milk[4-6]， and it was widely used in scientific research to guide the expression of foreign genes in milk. Parotidsecretoryprotein （PSP） is a protein specifically and highly expressed in saliva. The pig PSP gene is the best candidate gene for the development of pig parotid gland transgenic bioreactor and transgenic breeding research[7-8]. Sheep hair follicle keratin-associated protein 6.1 （KAP6.1） gene has been confirmed to be specifically expressed in sheep hair follicles[9]. In response to this feature， a series of hair follicle-specific expression vectors have been constructed using sheep KAP6.1 promoters[10-11].

The RELMβ gene has been confirmed to be specifically expressed in the intestinal tissues of rodents and humans[3]. Studies have shown that the RELMβ gene promoter region contains multiple intestinal epithelium-specific transcriptional regulatory factors， and the promoter region （-574-+215） fragment is the core region and contains key cis-acting elements[12]. The expression vector pcDNA3.1-RELMβ-XynB-Myc-GFP constructed in this study could effectively guide the specific expression of XynB in the intestinal cell line HT29 under the regulation of the intestine-specific expression promoter RELMβ. In the next step， in-vitro cell culture experiments will be carried out to determine the eukaryotic expression and secretion and enzyme activity of the constructed recombinant vector， as well as the stability of the secreted XynB enzyme， the optimal pH， etc.， to provide important materials for the realization of endogenous expression of XynB by transgenic technology.

References

[1] LIU WC， KIM IH. Effects of dietaryxylanase supplementation on performance and functional digestive parameters in broilers fed wheat-baseddiets[J]. Poultry Science， 2017， 96（3）： 566-573.

[2] NIE GX， WANG XQ， MING H， et al. Studies on the stability of xylanase produced by Aspergillus niger[J]. Acta Agriculturae Boreali-Sinica， 2004， 19（1）： 112-115. （in Chinese）

[3] STEPPAN CM， BAILEY ST， BHAT S， et al. Thehormone resistin links obesity to diabetes[J]. Nature， 2001， 409（6818）： 307-312.

[4] ARCHIBALD AL， MCCLENAGHAN M， HORNSEY V， et al. High-level expression of biologically active human alpha1-antitrypsin in the milk of transgenic mice[J]. Proceedings of the National Academy of Sciences， 1990， 87（13）： 5178-5182.

[5] NIAVARANI A， DEHGHANIZADEH S， ZEINALI S， et al. Development of transgenic mice expressing calcitoninasa β-lactoglobulin fusion protein in mammary gland[J].Transgenic Research， 2005， 14（5）： 719-727.

[6] BARASH I， FAERMAN A， RATOVITSKY T， et al. Ectopic expression of β-lactoglobulin/human serum albumin fusion genes in transgenic mice：hormonal regulation and in situ localization[J]. Transgenic Research， 1994， 3（3）： 141-151.

[7] YIN HF， FAN BL， YANG B， et al. Cloning of pigparotid secretory protein gene upstream promoter and the establishment of atransgenic mouse model expressing bacterial phytase for agricultural phosphorus pollution control[J]. Journal of Animal Science， 2006， 84（3）： 513-519.

[8] ZHANG XW， ZHANG GG， WU ZF， et al. Peptide linkers optimized recombinant enzyme gene of XynB-ManA and its co-expression in pK15 cells[J]. Scientia Agricultura Sinica， 2013， 46（22）： 4774-4783. （in Chinese）

[9] POWELL BC. The keratin protein and genes of wool and hair[J]. Wool Technology and Sheep Breeding， 1996， 44（2）： 100-118.

[10] WANG CS， AN TZ， BAI XJ， et al. Molecular cloning and activity analysis in sheep fibroblast of the keratin Associated protein promoter of the sheep[J]. Chinese Journal of Veterinary Science， 2008， 28（2）： 137-139， 145. （in Chinese）

[11] GUO XD， YIN J， YANG DS， et al. Construction of a hair-follicle-cell-specific expression vector of IGF-1 and its transfection into caprine fetal fibroblasts cells[J]. Acta Veterinaria et Zootechnica Sinica， 2009， 40（10）： 1460-1467. （in Chinese）

[12] CHEN Z， LEI MM， YU JN， et al. Cloning and sequence analysis of promoter region of porcine RELMβ gene[J]. Jiangsu Journal of Agricultural Sciences， 2015， 31（5）： 1060-1064. （in Chinese）