Study On Expressing Recombinant Human Lactoferrin By Silkworm Silkgland-bioreactor

Lactoferrin(LF) is an 80 kDa glycoprotein.Its protective effects range from direct antimicrobial activities against a large panel of microorganisms,including bacteria, viruses,fungi and parasites,to anti-inflammatory and anticancer activities.With the development of biotechnology,LF has been applicated in the fields of clinical medicine, food,Cosmetics and agriculture.But the traditional method of extracting LF can't afford the increasing market demand.Producing recombinant LF by transgenic technology is a good way to deal with the trouble.At present,there are various organisms could express LF including Saccharomyces cerevisiae,Spodoptera frugiperda,Rice,Bovine,Goat and so on.Creating new transgenic system which could express LF will be a supplementary of the front existing expressing systems and also will give us some new ideas and references.There are several advantages to develop silkworm as bioreactor.First,for thousands of years,people have been improving the silkworm lines in order to harvest more silk.The ability of secreting silk has been strengthened by artifical selection.One silkworm can produce 0.5g silk protein,which is far more than that produced by other kinds of spun silk insects.Second,as a relatively higher eukaryote,silkworm has the ability of protein modification.Third,the cost of feeding silkworm is very low.Totally, developing silkworm silkgland-bioreactor has enormous potential.Based on the above considerations,the study on expression of recombinant human lactoferrin by silkgland-bioreactor was carried out.1 Cloning of LF gene and constructing its transgenic expression vector Extract the total RNA of human breast and get the cDNA by reverse transcription. Amplify the full LF ORF fragments through PCR,and then clone the fragment in PMD19-T vector.The positive clone(PMD19-LF) was confirmed by sequencing. Digest PMD19-LF vector by restrictive enzyme BamHI and NotI.Purify the DNA fragment and ligate it with the vector(pSL[Serl-DsRed-SV40]) which was treated by BamHI and NotI,and then we got recombinant vector pSL[Serl-rhLF-SV40].Digest pSL[Serl-rhLF-SV40]by restrictive enzyme AscI and ligate the SerlrhLFSV40 fragment into the vector pBac[3xp3-EGFPafm]which was treated by AscI,constructing pBac[3xp3-EGFP,Serl-rhLF-SV40].2 Microinjection and screening of transformed animalsComicroinject the recombinant vector pBac[3xp3EGFP,Serl-rhLF-SV40]and helper vector pHA4PIG(proportion 1:1) into the silkworm DAZAO(P50) eggs which have been broken embryonic diapause.The final concentration of the DNA mixture is 400ng/ul.The total number of injected silkworm eggs is 772.Scan the generation 1 by fluorescence microscope and 9 positive broods including 94 positive individuals were obtained.3 Positive transgenic individual detected at molecular levelExtract the genomic DNA of the transgenic silkworm,then carry out Southern blotting and Inverse-PCR.The results showed that the hLF gene inserted into the silkworm genome and located on the chromosomes 5,6 and 9.The result also showed that there were at least three insertion patterns among the transgenic individuals obtained.RT-PCR showed that the Serl promoter could activate a high level transcriptive expression of hLF gene in silkgland.4 Extraction of recombinant protein and antibacterial activity analysisGrind the silkgland of 7^th day of 5^th instar larva in liquid nitrogen into powder and solve the powder in PBS,then take the supcrnatant after repeated freezing and thawing for several times.Gather the protein by affinity chromatography and ultrafiltration.The results from western blotting showed us that the rhLF was secreted by the silkgland. The test of anti-bacteria experiment showed that the rhLF could suppress the proliferation of E.coli and Staphylococcus aureus just like the standard sample of hLF, while the wild type silk-gland protein didn't show anti-bacteria activity.The results primarily showed us that the recombinant protein obtained in our study has the biological activity.