Construction Of Intestinal Stabilized Bovine Adenovirus And Its Accessment In Vitro

In recent years,much attention has been focused on the bovine adenovirus type 1(BAdV-1)and bovine adenovirus type 3(BAdV-3)due to their ability to gene delivery and other advantages,including lack of virulence,high transduction efficiency,high biosafety and act as the adjuvant.Currently,BAdV-3 has been widely used as vector for vaccine research and development.While the BAdV-3 is respiratory adenovirus,the main bottleneck of BAdV-3 as an oral vaccine vector is that BAdV-3 can not tolerate the extreme environment of the gastrointestinal tract.Although BAdV-1 is a type of gastrointestinal adenovirus and has good intestinal stability,the biology and vectored vaccine based on BAdV-1 was less reported.It is still no suitable bovine adenoviruses for oral vectored vaccine to date.In this work,we focused on two approaches to establish the intestinalstabilized bovine adenoviruses as vector for vaccine oral delivery basing on BAdV-3 and BAdV-1.1.We used the BJ5183 homologous recombination system to exchange the knob region at the top of the BAdV-3 fiber as the knob region at the top of the BAdV-1 fiber,and successfully rescued chimeric virus rBAdV3-B1 F with similar proliferative ability to wtBAdV3.We also found that the virus r BAdV3-B1 F could escape theBAdV3 neutralizing antibodies to a certain extent.However,recombinant virus rBAdV3-B1 F is also susceptible to low pH,pepsin,trypsin,and chymotrypsin similar to BAdV3.These results suggested that the chimeric adenovirus type 3 rBAdV3-B1 Fcould not improve the gastrointestinalstabilityby exchanging the knob region of fiber.2.We rescued recombinant virus rBAdV1-EYFP successfully by introducing the marker gene EYFP in E3 region of BAdV-1.PCR and Western blotting results showed that rBAdV1-EYFP could replicated continuously for serial passages.Neutralization test showed that the neutralization titer of rBAdV1-EYFP was consistent with wtBAdV1.Similar to wtBAdV1,the recombinant virus rBAdV1-EYFP could tolerate high levels of hydrochloric acid after treated in hydrochloric acid(pH=2).Moreover,the survival rate of recombinant virus rBAdV1-EYFP was higher than that of wild-type wt BAdV1 in the presence of pH,pepsin,trypsin and chymotrypsin.In this study,we attempted to construct the intestinal stabilized bovine adenovirus.By exchanging the knob region of the fiber protein of wtBAdV3,and it was found that the gastrointestinal stability of the recombinant virus did not improve,indicating that this method was not feasible.Furthermore,an recombinant bovine adenovirus type 1 with marker gene EYFP(rBAdV1-EYFP)was constructed for the first time.And we demonstratedthat the recombinant virus rBAdV1-EYFPcould tolerate high levels of hydrochloric acid,these results laid a foundation for developing bovine adenovirus vector vaccine with the stability of intestine.