| The silkworm, Bombyx mori, a well-known important economic insect, which occupies an important place in agriculture, industry and scientific research, has been domesticated and utilized for 5000 years in our country. Silk gland is a highly specialized tissue of silkworm, which synthesizes vast amounts of silk proteins and efficiently secretes them out to build a cocoon (about 0.5g per cocoon) in the late stage of 5 instar followed endomitosis. In view of the ability of efficient biosynthesis of silk proteins, plus the character of low cost and easy breeding, silkworm has been an ideal host for exogenous protein synthesis.For the past few years, the outbreak of Hand, foot and mouth disease (HFMD) seriously made a public health threat to the children in Asia-Pacific regions, especially in China. Now, there is no licensed specific medicine against HFMD yet. Therefore, urgent need to develop a wide-spectrum antigen, good immunogenicity, safe and stable vaccine, is the basic measure to prevent from this disease.We constructed a middle silk gland (MSG) spatiotemporal transgenic plasmid, which was based on the initial plasmid contained piggyBac transponsoaThe transgenic plasmid was micro-injected into the pre-blastoderm GO embryos.We obtained the transgenic germline after the genetic transformation and screening.To improve the efficiency of extraction and purification of exogenous protein, the sericin-like transgenic line was screened trough the transgenic line crossed with Nd-s strain.From the whole development stages and Inverse PCR result, we could indicate the transgene event did not influence the larval growth and development. The RT-PCR and QPCR results showed that VP1 gene was high expressed in the MSG of sericin-like transgenic larvae. SDS-PAGE and western blotting further proved that VP1 protein was successfully expressed in the transgenic silkworms. When we researched the extraction and purification conditions of VP1 protein, we firstly got that the best incubation time was 60 hours through the treatment of the sericin positive cocoons (SPCs) with different incubation time. Next, comparing with the concentrated normal positive cocoons (NPCs) of transgenic silkworms, results showed that we could reckon the contents of VP1 in each cocoon of SPCs and NPCs were 41.7ug and 9.8ug, respectively. Pure target VP1 proteins of SPCs and NPCs were obtained from total soluble proteins repeatedly concentrated and purified 3-5 times, which were firstly through 50kd and the filtrate followed by 30kd according to the size of target protein. And SDS-PAGE result showed that we could obtain about 500ng and 550ng VP1 from each SPC and NPC, with the purity of 90% and 70%, respectively. The VP1 antigen protein displayed the biological activity with polyclonal antibody EV71VLP and monoclonal antibody mD5 by ELISA assay. The above results demonstrated that the vaccine protein VP1 was successfully expressed in MSG and secreted into sericin layer of cocoon.To increase the expression of VP1 exogenous protein, the VP1 and red fluorescence protein expression cassettes were intended to site-specifically integrate into the translation initiation site of sericinl gene mediated by CRISPR/Cas9, which would untilize the promoter activity of endogenous serl and the efficient transcription of the endomitosis to utmostly increase the expression of exogenous gene. We obtained 20 and 8 chimeric pupae from two kinds of donor of single and dual sites at GO, which occupied 7.14% and 5.03% of total pupae, respectively. Among these pupae, gonad specific chimeras of two donors occupied 60% and 50% of total chimeras, respectively. It was a pity that the stable hereditary positive germline was not succeeded through genetic transformation. The result of cleavage efficiency by Cas9/sgRNAs showed both two sites had activity, but might be not enough for transformation of the positive germline. |
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