【摘要】：植物懸浮細(xì)胞具有良好的均一性、分散性、生長(zhǎng)迅速、生物價(jià)格低廉等特點(diǎn),因此利用植物懸浮系統(tǒng)作為植物反應(yīng)器生產(chǎn)生物藥,是極具價(jià)值與前景的項(xiàng)目。CD20分子為B淋巴細(xì)胞表面特有的抗原,CD20分子具有不易脫落、與抗體結(jié)合后不內(nèi)化、在人體血清中無(wú)游離形式存在等特征,因此是作為治療B細(xì)胞淋巴瘤的理想作用靶點(diǎn)而受到了人們的關(guān)注。本研究用紅花愈傷組織建立紅花懸浮細(xì)胞培養(yǎng)體系的條件,并通過(guò)農(nóng)桿菌介導(dǎo)轉(zhuǎn)化法利用紅花懸浮細(xì)胞表達(dá)CD20復(fù)合片段抗體。以紅花子葉為外植體,研究不同種類激素組合對(duì)愈傷誘導(dǎo)的影響,篩選優(yōu)質(zhì)愈傷組織進(jìn)行懸浮培養(yǎng),探索不同初始接種量、不同蔗糖質(zhì)量濃度、不同水解酪蛋白質(zhì)量濃度對(duì)紅花懸浮細(xì)胞系建立的影響。將CD20抗體基因兩端引入Xma I/EcoR I酶切位點(diǎn),并將其與pEASY-T1和pBasta載體相連,構(gòu)建pEASY-T1-CD20克隆載體與p Basta-CD20表達(dá)載體,并將構(gòu)建的pBasta-CD20表達(dá)載體轉(zhuǎn)入農(nóng)桿菌,利用農(nóng)桿菌介導(dǎo)紅花懸浮細(xì)胞轉(zhuǎn)化法表達(dá)CD20抗體。結(jié)果得出:紅花子葉愈傷誘導(dǎo)最佳條件為MS+NAA 2mg/L+6-BA 0.5mg/L、溫度(25±0.1)℃、光照70μmol/(m2·s)連續(xù)光照,繼代培養(yǎng)條件為MS+NAA 1mg/L+6-BA 0.5mg/L,30μmol/(m2·s)連續(xù)光照,溫度(25±0.1)℃。懸浮細(xì)胞體系培養(yǎng)最佳條件:不加瓊脂粉的MS+NAA1mg/L+6-BA 0.5mg/L、接種量2.5g/50mL、蔗糖濃度20g/L、水解酪蛋白濃度800mg/L。結(jié)論:pBasta-CD20表達(dá)載體構(gòu)建成功且目的蛋白在紅花懸浮細(xì)胞成功表達(dá),成功建立了紅花懸浮細(xì)胞培養(yǎng)體系,并成功用該體系表達(dá)了CD20抗體,為利用懸浮細(xì)胞生產(chǎn)CD20抗體提供了新途徑。
[Abstract]:Plant suspension cells have good uniformity, dispersity, rapid growth and low biological cost, so plant suspension system is used as plant reactor to produce biological drugs. CD20 molecule is a specific antigen on the surface of B lymphocytes, and CD20 molecule is not easy to fall off, does not internalize after binding with antibody, and has no free form in human serum. Therefore, as an ideal target for the treatment of B cell lymphoma, people pay more attention to it. In this study, the culture system of safflower suspension cells was established by callus of safflower, and CD20 antibody was expressed by Agrobacterium tumefaciens mediated transformation. The effects of different hormone combinations on callus induction were studied with the cotyledon of safflower as explants. The high quality callus was selected for suspension culture, and different initial inoculation amount and different sucrose concentration were explored. Effects of different concentration of hydrolyzed casein on the establishment of safflower suspension cell line. The two ends of CD20 antibody gene were introduced into the Xma I/EcoR I restriction site and connected with pEASY-T1 and pBasta vectors to construct pEASY-T1-CD20 clone vector and p Basta-CD20 expression vector. The constructed pBasta-CD20 expression vector was transformed into Agrobacterium tumefaciens. CD20 antibody was expressed by Agrobacterium tumefaciens mediated safflower suspension cell transformation. The results showed that the optimum conditions for callus induction in cotyledons of safflower were: MS NAA 2mg/L 6-BA 0.5 mg / L, temperature (25 鹵0.1) 鈩，

本文編號(hào)：2295247