Adenovrial Vector Mediates High Expression Levels Of Human Lactoferrin In The Milk Of Rabbits And Goats

The mammary gland has been identified as a suitable alternative to produce biopharmaceutical and nutritional proteins, which are synthesized in the mammary epithelial cells and thereafter secreted into the milk from which the purification process is relatively simple. So, transgenesis has been extensively used for the genetic modification of farm animals to produce human recombinant proteins in milk. This system concluding four steps:(i)First,construct the recombinant expression vectors and special sequences are specific for mammary glad expression; But it is known that this control system is not absolute, allowing for transgene expression founders might die even in the fetal stage, where only the animals in which the expression levels were very low were born. (ii) Through some transgene methods and get the recombinant embryo or cells. (iii) Embryo transfer and nuclear transfer and got the transgene animal ;(vi) after breeding the transgene animals, which to be detected for expression target gene.However, drawbacks in this technology include (i) the technically difficult procedure required, its low efficiency in produce transgenic animals;(ii) especially its inefficiency when applied to large livestock, (iii)the length of time between incorporating foreign DNA and harvesting the exogenous protein,(vi)and the health risk for animals due to the ectopic expression of transgenes. The direct in vivo transfection of the mammary gland has been proposed as a faster and more inexpensive alternative to target the expression of a heterologous gene to the secretory mammary epithelial cells. Although these expression levels may be enough for certain basic studies, they must be improved when large amounts of recombinant proteins are required.Build on above traditionally technology system shortcomings,this paper reported a strategy to produce transgenic recombinant protein in the milk of animal mammary gland, such as rabbits and goats. Adenoviral vectors are able to successfully infect the mammary glandular epithelium and to promote the secretion of high amounts of a recombinant protein in the milk of animals. This system has some merits (i) the direct transduction of the mammary gland with adenoviral vectors could be a very useful tool to study the powerfulness of mammary-specific promoters avoiding variables associated to the position effects or to the copy numbers. The use of constitutive promoters, undoubtedly simplifies vector design, however this does not preclude the use of tissue specific promoters for special applications. (ii) It is possible to obtain high expression levels of a foreign protein in milk. (iii) The mammary gland can produce an in vivo biologically active recombinant protein. (vi) The secretion of the recombinant human lactoferrin in the milk is not permanence expression, so have low products cost.Members of the adenovirus family infect a great variety of post-mitotic cells; even those associated with highly differentiated tissues such as skeletal muscle, lung, brain and heart. Since they deliver their genome to the nucleus and can replicate with high efficiency, they are prime candidates for the expression and delivery of therapeutic genes. They have a wide host-range and are currently divided into three genera with further subdivision into species (also termed subgenera or subgroups) A to F. Division of human stereotypes, based mainly on immunological criteria, has historically been the basis of classificationRecombinant adenoviruses currently are used for a variety of purposes, including gene transfer in vitro, vaccination in vivo, and gene therapy. Several features of adenovirus biology have made such viruses the vectors of choice for certain of these applications. For example, adenoviruses transfer genes to a broad spectrum of cell types, and gene transfer is not dependent on active cell division. Additionally, high titers of viruses and high levels of transgene expression generally can be obtained. The ability of adenovirus vectors to deliver and express genes at high yields, especially in vitro, however, to date, there has little reports have been used adenovirus for production of transgenic recombinant protein. The objective of our study is to investigate a new method by using recombinant adenovirus, which can produce the biopharmaceutical, and nutritional proteins, which are synthesized in the mammary epithelial cells and thereafter secreted into the milk. The method may be used also to assess the functionality of gene constructs prior to the generation of a transgenic animal, and may be a viable approach to study the physical, chemical and biological characteristics of recombinant proteins expressed in milk of different species at the same time.In order to establish a platform for the development of new biotechnological processes directed to the production of recombinants complex proteins in milk. We have carried out the studies on the cloning of objective gene and construction of mammalian expression vector and recombinant adenovirus containing hLTF cDNA, also, the rAd vectors direct transduction of the mammary gland. The main results are as following:1. Human lactoferrin (hLTF) cDNA was amplified by RT-PCR from normal human mammary tissue, and then subcloned into pcDNA3.1 vector. hLTF cDNA sequence was determined, which consists of 2169bp. Comparison with five other hLTF cDNA sequences registered in GeneBank shows 99 % homology in DNA sequence. Four mammalian expression vectors were constructed, the pLG and p3LG vectors have GFP report gene and the incorporation of a GFP protein in this vector was designed to serve as a live marker for tracking infected cells or tissues in animal studies. The vectors p3LF and pDCLF use the neomycin resistance gene (Neor) to permit selection of transformed cells. The pLG plasmid and p3LG vector use the internal ribosome entry site from the encephalomyocarditis virus, which allows translation of two consecutive open reading frames from the same messenger RNA.2. The recombinant eukaryotic expression vector p3LF, p3LG, pDCLF containing human lactoferrin gene cDNA was transfected into COS7 cells, Chinese hamster ovary (CHO) cells and Human embryonic kidney cells (HEK293) cells by Lipofectamine transinfection. Positive single clone cells were selected with G418 and by PCR. The result of Western blotting analysis on cultured cell supernatant shows that transfected cells can express the exogenic gene and secrete hLTF protein. Analyses of expression efficiencies of selected G418-resistant clones showed that the expression of hLTF mRNA and recombinant protein were higher in CHO and HEK293 cells than those in COS7 cells. But no significant difference between the three cells lines in expression protein. However, the COS7 are suitable for transient expression target recombinant protein. The result of antibacterial experiment indicates that eukaryotic expression vector p3LF, p3LG, pDCLF containing human lactoferrin gene cDNA transfection into the mammalian cells CHO, HEK293 cells and COS7 cells and these cells can expression human lactoferrin in high efficiency, which provides the basis for further research of recombinant human lactoferrin protein in mammalian. The recombinant hLTF protein has the effect of inhibiting E. coli proliferation; moreover, its activity is superior to the commercial available hLTF standard proteins.3. To obtain the recombinant adenoviruses, we developed a system that simplifies the generation and production of adenoviruses containing human lactoferrin transgene in bacteria. The hLTF cDNA was obtained from above steps and was sub cloned into a shuttle vector. After sequence confirmation, the resultant plasmid was linearized by the restriction endonulease Pme co and I transformed with the super coiled adenoviral vector pAdeasy into Escherichia coli strain BJ5183. Recombinants were confirmed by colony PCR and restriction endonulease digestion. The adenoviral vector Ad-LTF was propagated in HEK 293 cells and purified by Cesium chloride density centrifugation. PCR and Western blot analysis were performed to confirm hLTF expression. Verification of viral genome is performed and expression of transgene is evaluated in 293 cells and CHO cells for identity. For adenovirus carrying the human hLTF transgene, quantities hLTF expression is demonstrated by PCR and Western blot assay following infection of CHO cells. The results suggestion, recombinant adenoviruses can be simple generated by using modified Adeasy system results in highly efficient viral production in Escherichia coil. Ad-hLTF can be further used in in vivo gene therapy studies and the production of recombinant proteins of biopharmaceutical interest.4. The Cre-lox recombination system ahs the potential to become a powerful tool the conditional and cell-specific deletion of genes. The introduction of this system into produces the recombinant adenovirus. First, the hLTF cDNA fragment was cloned into plasmid pDC311 vector, and after sequence confirmation, the resultant plasmid and the plasmid pBHGlox (delta) E1, 3Cre co-transfection on HEK 293 cells. PCR and Western blot analysis were performed to confirm hLTF expression. Verification of viral genome is performed and expression of transgene is evaluated in 293 cells and CHO cells for identity. For adenovirus carrying the human hLTF transgene, quantities hLTF expression is demonstrated by PCR and Western blot assay following infection of CHO cells. The results suggestion, recombinant adenoviruses can be simple generated by using Cre-lox system results in highly efficient viral production in HEK 293 cells. Ad-LTF can be further used in vivo gene therapy studies and the production of recombinant proteins of biopharmaceutical interest.5. The recombinant adenovirus containing human lactoferrin cDNA was amplified in HEK293 for four times. Then the final title of recombinant adenovirus was purified. A direct instillation of a recombinant adenoviral vector containing an expression cassette for the human lactoferrin into the mammary gland of rabbits and goats allowed for the efficient secretion of human lactoferrin in the milk. Western blot analysis were performed to confirm hLTF expression after the adenovirus infection on 48h up to 10 d. Through this approach we were able to express human lactoferrin high levels in the milk of rabbits and goat milk and the recombinant protein expression up to 7-10d. This technology platform still to be improved to performed in transgene protein production in the milk of animals.Conclusions: (1) Human lactoferrin (hLTF) cDNA was amplified by RT-PCR from normal human mammary tissue and four mammalian expression vectors were constructed successfully. (2) The recombinant eukaryotic expression vector p3LF, p3LG, pDCLF containing human lactoferrin gene cDNA was transfected into COS7 cells, Chinese hamster ovary (CHO) cells and Human embryonic kidney cells (HEK293) cells by Lipofectamine transinfection. These cells can expression human lactoferrin in high efficiency. (3) We developed a method of produce the Ad-hLTF recombinant adenoviruses by using modified Adeasy system results in highly efficient viral production in Escherichia coil. Ad-hLTF can be further used in in vivo gene therapy studies and the production of recombinant proteins of biopharmaceutical interest. (4) We established a method for produce the Ad-hLTF recombinant adenoviruses by using Cre-lox system in mammalian cells. These modifications result in highly efficient viral production systems, which can be further used to produce any recombinant adenovirus containing target gene. (5) We obtained high levels expression functional recombinant hLTF protein in the milk of rabbits and goats.