Expression of recombinant human butyrylcholinesterase in the milk of transgenic mice

doi:10.15302/J-FASE-2014020

Front. Agr. Sci. Eng.

2014, Vol. 1

Issue (3) : 179-184 https://doi.org/10.15302/J-FASE-2014020

LETTER

Expression of recombinant human butyrylcholinesterase in the milk of transgenic mice

Dan LU¹,Shengzhe SHANG¹,Shen LIU¹,Ying WU¹,Fangfang WU²,Tan TAN¹,Qiuyan LI¹,Yunping DAI¹,Xiaoxiang HU¹,Yaofeng ZHAO¹,Ning LI^1,^*(

)

1. State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing100193, China
2. College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650000, China

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Abstract

Butyrylcholinesterase (BCHE) is a natural bioscavenger that protects humans against organophosphate toxicity. Due to the limited yield of human BCHE (hBCHE) when purifying from human plasma, it is necessary to find an alternative method to produce this protein. One potential method is to produce transgenic livestock that make modified milk containing high concentration of hBCHE. In this study, we cloned the hBCHEgene into a human lactoferrin (hLF) bacterial artificial chromosome (BAC) construct to make a hLF-hBCHE BAC construct. Subsequently, we injected the BAC construct into pronuclei of mouse fertilized embryos and generated transgenic mice. Expression analysis showed that recombinant hBCHE (rhBCHE) was expressed efficiently in the mammary gland of the transgenic mice and the concentration of rhBCHE in the milk of individual mice ranged from 76±12 to 159±28 mg·L^-1. Protein function tests showed that rhBCHE has the same enzymatic activity as the native hBCHE. Our results pave the way for making transgenic livestock to produce large quantities of rhBCHE.

Keywords recombinant human butyrylcholinesterase (rhBCHE) human lactoferrin bacterial artificial chromosome (hLF BAC) transgenic mice milk

Corresponding Author(s): Ning LI

Online First Date: 03 December 2014 Issue Date: 27 January 2015

Cite this article:

Dan LU,Shengzhe SHANG,Shen LIU, et al. Expression of recombinant human butyrylcholinesterase in the milk of transgenic mice[J]. Front. Agr. Sci. Eng. , 2014, 1(3): 179-184.

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https://academic.hep.com.cn/fase/EN/10.15302/J-FASE-2014020
https://academic.hep.com.cn/fase/EN/Y2014/V1/I3/179

Fig.1 Sketch of the construction of the pBAC-hLF-hBCHE-Neo expression plasmid. The construction of the pBAC-hLF-hBCHE-Neo expression plasmid was divided into three distinct steps. Step 1: a 2 kb kanamycin/neomycin resistance cassette flanked by two homologous arms was obtained by PCR and electroporated into competent SW102 cells containing hBCHE BAC to generate the hBCHE minigene BAC; Step 2: arms H1 and H2 were dissociated by Not I digestion to obtain the 20 kb hBCHE mini genomic coding sequence from ATG to TAA; Step 3: the 29 kb hLF genomic sequence was replaced with 20 kbhBCHE mini genomic DNA on the hLF BAC. The pBAC-hLF-hBCHE-Neo construct contains a 95 kb 5′ flanking region of the hLF gene, the 20 kb hBCHE mini genomic fragment with a kanamycin/neomycin resistance cassette for future SCNT and a 30 kb 3′ flanking region of the hLFgene.

Tab.1 Expression of recombinant human butyrylcholinesterase in the milk of transgenic mice

Fig.2 Molecular characterization of transgenic mice. (a) PCR detection of transgenic founders. M: 100bp DNA ladder; PC: positive control using pBAC-hLF-hBCHE-Neo construct; NC: negative control; lane 1-12;transgenic founders numbered from 1 to12; (b) Southern blot analysis of transgenic founders. Digested genomic DNA of two transgenic founders (F₀-4 and F₀-5), one F₁ transgenic mouse from founder line F₀-4 (F₁-7) and non-transgenic mice. An expected 2.9 kb band was detected in transgenic samples. B: blank; NC: genomic DNA of non-transgenic mice as a negative control; P1, P2, P3 represent 1, 5 and 10 copies of pBAC-hLF-hBCHE-Neo plasmid, respectively; (c) RT-PCR analysis of transgene expression in transgenic mouse tissues. All tissue RNAs used for RT-PCR were collected during the middle of the lactation period. Mouse GAPDH was used as the RT-PCR internal control. M: 100bp DNA ladder; WT: wild type mouse mammary gland; MG: transgenic mouse mammary gland; H: heart; Li: liver; Sp: spleen; Lu: lung; K: kidney; St: stomach; I: intestine; Mu: muscle; (d) Western blot analysis of recombinant human butyrylcholinesterase (rhBCHE) expression in milk of transgenic mice. The milk samples were collected during the middle of the lactation period, separated by SDS-PAGE and then transferred to nitrocellulose membranes. NC: milk from non-transgenic mice (negative control); PC: hBCHE standard (positive control); F₀-4 and F₀-5: diluted milk (1:3) from transgenic founders numbered 4 and 5; (e) non-denaturing gel stained for BCHE activity. PC1: purified plasma hBCHE (2.5 μL); PC2: purified plasma hBCHE (1μL); NC: diluted non-transgenic mouse milk (1:3); F₀-4, F₁-7, F₁-9 and F₁-14: diluted transgenic mice milk (1:3); 2 mer: dimer; 4 mer: tetramer.

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