Construction Of PreS1Infection Determinant Randomized HBV Bank And Its Application In Small Animal

The hepatitis B virus (HBV) belongs to hepadnaviruses, whichcharacteristically display a hepatic tropism and a restricted host range thatextends only to closely related species. Currently, the main animal models forstudy of HBV infection are chimpanzee, tree shrew, woodchuck, duck and avariety of mouse models. But because of shortage of sources, high price, lowinfection efficiency, instability of the experimental results, low rate ofreproduction, ferocious behavior, difficulty in preparation of model, orinability of fully repeating of HBV infection cycle and so on, theirapplications are limited. Compensating these shortcomings and building newanimal and cell models of HBV are hot and difficult at present.Plenty of evidences from previous researches indicated that PreS1domain of HBV was directly involved in HBV entry to hepatocyte andcontribute to host specificity and tissue tropism. Studies mapped the minimalactivity domain of pre-S1for HBV infection to residues5to20. According tothe data, we randomized the aa9-17, aa12-21and aa16-24residues of PreS1domain respectively by recombinant DNA technology, resulting in three PreS1randomized HBV banks, and study their infectivity of small convenientanimal. The study was composed of two parts as follows.Part1Construction of PreS1randomized HBV bankObjective: To randomize the aa9-17, aa12-21and aa16-24residues ofPreS1domain respectively, and constructed three PreS1randomized HBVbanks, using of which to filter HBV susceptible animal model.Methods:1Based on plasmid pCH-3093, we constructed three transitional plasmids pCH-3093-Alw(-), pCH-3093-AlwNl and pCH-3093-Insert one by one.2Designed and synthesized three randomized primers of PreS1domain S1rand9-17, S1rand12-21and S1rand16-24, and the universal extensionprimer S11038(-). Used the three randomized primers and the extensionprimer synthesize randomized fragments respectively with Klenow Fragment.3Inserted the three randomized fragments into the corresponding position ofwild type HBV expression plasmid pCH-3093, and constructed three PreS1randomized HBV banks respectively.4Determined the capacity, randomness and replication efficacy of the threePreS1randomized HBV banks.Result:1The three transitional plasmids pCH-3093-Alw(-), pCH-3093-AlwNl andpCH-3093-Insert were constructed successfully.2The three randomized primers S1rand9-17, S1rand12-21and S1rand16-24, and the primer S11038(-) were synthesized. The three randomizedfragments were constructed and followed by three PreS1randomized HBVbanks successfully.4Statistical analyses showed that capacity of the PreS1randomized HBVbanks was approximately107, and the actual distribution of amino acids hadno significant differences with theoretical frequency. We got a great quantityof highly purified plasmid. Southern Blotting detection confirmed that thereplication capacity of the three random banks were close to each other, butweaker than wild-type HBV.Conclusion: We constructed three PreS1randomized HBV bankspCH-S1-917, pCH-S1-1221and pCH-S1-1624, whose capacity andrandomness were satisfactory. Their replication efficacy was similar to eachother, but weaker than wild-type HBV, which was considered to be associatedwith base variation. However it didn’t affect the application of randomizedHBV banks. The three PreS1randomized HBV banks provide a convenientexperimental tool to explore the corresponding HBV animal models. Part2Filter HBV animal model by PreS1randomized HBV banksObjective: To filter HBV animal model and map the region of PreS1domain involving in HBV attachment to hepatocyte through Wistar rat,Sprague Dawley rat, BALB/C mice and New Zealand White Rabbit infectedby PreS1randomized HBV banks.Methods:1Wistar rat, Sprague Dawley rat, BALB/C mice and New Zealand WhiteRabbit were injected with the three PreS1randomized HBV banks and takenblood from them after one and two months later to test the serum HBV-DNA.2Two month after injection, sacrificed the animala and isolated HBV cccDNAwith the animal liver, from which PreS1randomized domain was amplified byPCR, and then was re-cloned to HBV plasmid to construct the second roundPreS1randomized HBV bank and measured the bank sequence.3Injected animals with the second round round PreS1randomized HBV bankand test the serum HBV-DNA one and two month later.4Two month after injection, detected the HBsAg and HBcAg expression ofliver from animals infected with PreS1randomized HBV banks byimmunohistochemistry.Result:1After infected with the PreS1randomized HBV bank, animals’ health werein good condition. One month after injection, serum HBV-DNA was positivefor all animals injected with randomized HBV banks. Wistar rat (W7) had themost viral titer of2.43×105copies/mL, and the others were between103and104copies/mL. However, the animals injected with wild type HBV were allnegative.2HBV cccDNA could be extracted from rabbits and BALB/C mouse, whileWistar rats, Sprague Dawley rats and control group were all negative. Possiblythe viral had been cleared in Wistar rat and Sprague Dawley rats.3The second round preS1randomized HBV banks pCH-S1-rabbit andpCH-S1-mouse were constructed successfully through inserting the PreS1 randomized fragment into the corresponding position of wild type HBV.4sequence measurement of the second round PreS1randomized HBV banksshowed that pCH-S1-rabbit came from pCH-S1-1221completely, while as forpCH-S1-mice,15/16came from pCH-S1-1221, and1/16was frompCH-S1-917.5One and two month after Rabbits and BALB/C mice injected with thesecond round PreS1randomized HBV banks pCH-S1-rabbit andpCH-S1-mouse respectively, serum HBV-DNA were all weakly positive.62month after injection, Immunohistochemistry showed that HBsAg andHBcAg expressed in all animals either injected with the first roundrandomized or the second round. However, Wistar rat (W7) had the highestexpression level, and the main distribution of HBsAg and HBcAg distributedboth in and out of cell.Conclusion: Wistar rat, Sprague Dawley rat, BALB/C mice and NewZealand White Rabbit could be infected by the PreS1randomized HBV banks,and serum HBV-DNA was positive. Both HBsAg and HBcAg expressed inanimal livers. Sequence measurement of PreS1randomized fragmentextracted from liver certified that aa12-21residues of PreS1domain isessential in HBV infection, and affect HBV host specificity To a certain extent.However, infection caused by the random banks is a acute process. It needs tobe transformed further to suppress the host immune response at the gene level,and we will get a ideal chronic infection HBV animal model.